Analogs of ribosome-inactivating proteins

ABSTRACT

The present invention provides purified and isolated polynucleotides encoding Type I ribosome-inactivating proteins and analogs thereof having a cysteine available for disulfide bonding to targeting molecules. Vectors comprising the polynucleotides and host cells transformed with the vectors are also provided. Preferred analogs according to the present invention are analogs of Type I ribosome-inactivating proteins (1) having a cysteine available for intermolecular disulfide bonding located at an amino acid position corresponding to a position not naturally available for intermolecular disulfide bonding in the Type I ribosome-inactivating protein and corresponding to a position on the surface of ricin A-chain in its natural conformation and (2) retaining ribosome-inactivating activity of the Type I ribosome-inactivating protein. The RIP analogs are particularly suited for use as components of cytotoxic therapeutic agents and, more specifically, as components of immunotoxins. Cytotoxic agents according to the present invention may be used to selectively eliminate any cell type to which the RIP component is targeted by the specific binding capacity of the second component, and are suited for treatment of diseases where the elimination of a particular cell type is a goal, such as autoimmune disease, cancer and graft-versus-host disease.

This application is a continuation-in-part of U.S. patent applicationSer. No. 07/787,567 filed on Nov. 4, 1991, now abandoned.

BACKGROUND

The present invention relates, in general, to materials useful ascomponents of cytotoxic therapeutic agents. More particularly, theinvention relates to analogs of ribosome-inactivating proteinsspecifically modified for conjugation to targeting molecules and topolynucleotides encoding ribosome-inactivating proteins and analogsthereof.

Ribosome-inactivating proteins (RIPs) comprise a class of proteins whichis ubiquitous in higher plants. RIPs have also been isolated frombacteria. RIPs are potent inhibitors of eukaryotic protein synthesis.The N-glycosidic bond of a specific adenine base is hydrolyticallycleaved by RIPs in a highly conserved loop region of the 28S rRNA ofeukaryotic ribosomes thereby inactivating translation.

Stirpe et al., FEBS Lett., 195(1,2), 1-8 (1986) groups plant RIPs intotwo types. Type I proteins each consist of a single peptide chain havingribosome-inactivating activity, while Type II proteins each consist ofan A-chain, essentially equivalent to a Type I protein, disulfide-linkedto a B-chain having cell-binding properties. Gelonin, dodecandrin,tricosanthin, tricokirin, bryodin, Mirabilis antiviral protein (MAP),barley ribosome-inactivating protein (BRIP), pokeweed antiviral proteins(PAPs), saporins, luffins and momordins are examples of Type I RIPs,while ricin and abrin are examples of Type II RIPs. Amino acid sequenceinformation is reported for various ribosome-inactivating proteins. Itappears that at least the tertiary structure of active sites isconserved among Type I RIPs, bacterial RIPs and A-chains of Type II RIPsand, in many cases, primary structure homology is also found. Ready etal., J. Biol. Chem., 259(24), 15252-15256 (1984) and other reportssuggest that the two types of RIPs are evolutionarily related.

Separated from their natural environment, Type I plantribosome-inactivating proteins may be particularly suited for use ascomponents of cytotoxic therapeutic agents. A RIP may be conjugated to atargeting agent that will deliver the RIP to a particular cell type invivo in order to selectively kill those cells. Because some RIPs, suchas the Type I RIP gelonin, are only available from scarce plantmaterials, it is desirable to clone the genes encoding the RIPs toenable recombinant production of the proteins. It is also desirable todevelop analogs of the natural proteins which may be easily conjugatedto targeting molecules while retaining their natural biological activitybecause most Type I RIPs have no natural sites (i.e. available cysteineresidues) for conjugation to targeting agents.

There thus exists a need in the art for cloned genes encodingribosome-inactivating proteins and for analogs of ribosome-inactivatingproteins which may be easily conjugated to targeting molecules.

SUMMARY OF THE INVENTION

The present invention provides purified and isolated polynucleotidesencoding analogs of Type I ribosome-inactivating proteins having acysteine available for disulfide bonding to targeting molecules. Vectorscomprising the polynucleotides and host cells transformed with thevectors are also provided.

Analogs of a Type I plant RIP are defined herein as non-naturallyoccurring polypeptides that share the ribosome-inactivating activity ofthe natural protein but that differ in amino acid sequence from thenatural protein. Preferred analogs according to the present inventionare analogs of Type I plant RIPs each having a cysteine available fordisulfide bonding located at a position in its amino acid sequence fromthe position corresponding to position 251 in SEQ ID NO: 1 to thecarboxyl terminal position of the analog. Other preferred analogsaccording to the invention are Type I RIPs each having a cysteineavailable for disulfide bonding at a position in the analog that is onthe surface of the protein in its natural conformation and that does notimpair native folding or biological activity of theribosome-inactivating protein. Analogs of bacterial RIPs are alsocontemplated by the present invention.

The present invention provides an analog of a Type Iribosome-inactivating protein, which analog has a cysteine available forintermolecular disulfide bonding at an amino acid position correspondingto a position not naturally available for intermolecular disulfidebonding in the Type I ribosome-inactivating protein and which cysteineis located at a position in the amino acid sequence of the analogcorresponding to position 259 in SEQ ID No: 1 or at a position in theamino acid sequence in the analog corresponding to a position from theposition corresponding to position 251 in SEQ ID NO: 1 to the carboxylterminal position of the analog.

An analog according to the present invention may be an analog ofgelonin. In an analog of gelonin according to the present invention, thecysteine may be at a position in the analog from position 244 to thecarboxyl terminal position of the analog, more preferably at a positionin the analog from position 247 to the carboxyl terminal position of theanalog, and, in these regions, most preferably at position 244, atposition 247 or at position 248 of the amino acid sequence of theanalog. It is preferred that the gelonin cysteine residues at positions44 and 50 be replaced with alanine residues.

An analog according to the present invention may be an analog of barleyribosome-inactivating protein. Preferably, a cysteine in such an analogis at a position in the analog from position 256 to the carboxylterminal position, and more preferably the cysteine is at a position inthe amino acid sequence of the analog from position 260 to the carboxylterminal position of the analog. Most preferably, in these regions, thecysteine is at position 256, at position 270 or at position 277 of theamino acid sequence of the analog.

An analog according to the present invention may be an analog ofmomordin II.

Analogs according to the present invention may have a cysteine in theamino acid sequence of the analog at a position which corresponds to aposition within one amino acid of position 259 of SEQ ID NO: 1. Such ananalog may be an analog of gelonin, of barley ribosome-inactivatingprotein, or of momordin II.

The present invention also provides a polynucleotide encoding an analogof a Type I ribosome-inactivating protein, which analog has a cysteineavailable for intermolecular disulfide bonding at an amino acid positioncorresponding to a position not naturally available for intermoleculardisulfide bonding in the Type I ribosome-inactivating protein, and whichcysteine is located at a position in the amino acid sequence of theanalog from the position corresponding to position 251 in SEQ ID NO: 1to the carboxyl terminal position of the analog. The polynucleotide mayencode an analog of gelonin, preferably an analog wherein the cysteineis at a position in the amino acid sequence of the analog from position244 to the carboxyl terminal position of the analog, more preferablywherein the cysteine is at a position in the analog from position 247 tothe carboxyl terminal position of the analog, and most preferably thecysteine is at position 244, at position 247 or at position 248 of theamino acid sequence of the analog. It is preferred that a polynucleotideaccording to the present invention encode a gelonin analog wherein thenative gelonin cysteine residues at positions 44 and 50 are replacedwith alanine residues.

A polynucleotide according to the present invention may encode an analogof barley ribosome-inactivating protein, preferably an analog whereinthe cysteine is at a position in the analog from position 256 to thecarboxyl terminal position of the analog, more preferably wherein thecysteine is at a position in the analog from position 260 to thecarboxyl terminal position of the analog, and most preferably whereinthe cysteine is at position 256, at position 270 or at position 277 ofthe amino acid sequence of the analog.

A polynucleotide according to the present invention may encode an analogof mormordin II.

The present invention provides a vector including a polynucleotideencoding an analog of a Type I ribosome-inactivating protein, whichanalog has a cysteine available for intermolecular disulfide bonding ata amino acid position corresponding to a position not naturallyavailable for intermolecular disulfide bonding in the Type Iribosome-inactivating protein and which cysteine is located at aposition in the amino acid sequence of the analog from the positioncorresponding to position 251 in SEQ ID NO: 1 to the carboxyl terminalposition of the analog.

The present invention further provides a host cell including a DNAvector encoding an analog of a Type I ribosome-inactivating protein,which analog has a cysteine available for intermolecular disulfidebonding at an amino acid position corresponding to a position notnaturally available for intermolecular disulfide bonding in the Type Iribosome-inactivating protein and which cysteine is located at aposition in the amino acid sequence of the analog from the positioncorresponding to position 251 in SEQ ID NO: 1 to the carboxyl terminalposition of the analog. In such a host cell the vector may encode ananalog of gelonin, especially an analog wherein the cysteine is atposition 247 of the amino acid sequence of the analog, such as in thehost cell deposited as A.T.C.C. Accession No. 69009.

A host cell according to the present invention may include a vectorencoding barley ribosome-inactivating protein, especially a host cellwherein the cysteine is at position 277 of the amino acid sequence ofthe analog such as in the host cell deposited as A.T.C.C. Accession No.68722.

The present invention also provides an agent toxic to a cell includingan analog of a Type I ribosome-inactivating protein linked by adisulfide bond through a cysteine to a molecule which specifically bindsto the cell, which cysteine is at an amino acid position in the analogcorresponding to a position not naturally available for intermoleculardisulfide bonding in the Type I ribosome-inactivating protein and whichcysteine is located in the amino acid sequence of the analog from theposition corresponding to position 251 in SEQ ID NO: 1 to the carboxylterminal position of the analog. The agent may include an analog ofgelonin, preferably an analog wherein the cysteine is at a position inthe analog from position 247 to the carboxyl terminal position of theanalog, and more preferably wherein the cysteine is at position 247 or248 of the amino acid sequence of analog. An agent including an analogwherein the native gelonin cysteine residues at positions 44 and 50 arereplaced with alanine residues is preferred.

An agent according to the present invention may include an analog ofbarley ribosome-inactivating protein, preferably an analog wherein thecysteine is at a position in the analog from position 260 to thecarboxyl terminal position of the analog, more preferably wherein thecysteine is at a position in the analog from position 270 to thecarboxyl terminal position of the analog, and most preferably whereinthe cysteine is at position 256, at position 270 or at position 277 ofthe amino acid sequence of the analog.

An agent according to the present invention may include an analog ofmomordin II.

The present invention provides an agent wherein the Type Iribosome-inactivating protein is linked to an antibody, particularly toan H65 antibody or to an antibody fragment, more particularly to anantibody fragment selected from the group consisting of chimeric andhuman engineered antibody fragments, and most particularly to a Fabantibody fragment, a Fab' antibody fragment or a F(ab')₂ antibodyfragment. It is highly preferred that an agent according to the presentinvention include a chimeric or human engineered antibody fragmentselected from the group consisting of a Fab antibody fragment, a Fab'antibody fragment and a F(ab')₂ antibody fragment.

A method according to the present invention for preparing an analog of aType I ribosome-inactivating protein includes the step of expressing ina suitable host cell a polynucleotide encoding a Type Iribosome-inactivating protein having a cysteine available forintermolecular disulfide bonding substituted (e.g., by site-directedmutagenesis of the natural DNA sequence encoding the RIP or by chemicalsynthesis of a DAN sequence encoding the RIP analog) at an amino acidposition corresponding to a position not naturally available forintermolecular disulfide bonding in the Type I ribosome-inactivatingprotein, which cysteine is located at a position in the amino acidsequence of the analog from the position corresponding to position 251in SEQ ID NO: 1 to the carboxyl terminal position of the analog.

A product according to the present invention may be a product of amethod including the step of expressing in a suitable host cell apolynucleotide encoding a Type I ribosome-inactivating protein having acysteine available for intermolecular disulfide bonding substituted atan amino acid position corresponding to a position not naturallyavailable for intermolecular disulfide bonding in the Type Iribosome-inactivating protein, which cysteine is located at a positionin the amino acid sequence of the analog from the position correspondingto position 251 in SEQ ID NO: 1 to the carboxyl terminal position of theanalog.

The present invention provides a method for preparing an agent toxic toa cell including the step of linking an analog of a Type Iribosome-inactivating protein through a cysteine to a molecule whichspecifically binds to the cell, which analog has the cysteine at anamino acid position corresponding to a position not naturally availablefor intermolecular disulfide bonding in the Type I ribosome-inactivatingprotein and which cysteine is located at a position in the amino acidsequence of the analog from the position corresponding to position 251in SEQ ID NO: 1 to the carboxyl terminal position of the analog.

According to the present invention, a method for treating a disease inwhich elimination of particular cells is a goa_(I) may include the stepof administering to a patient having the disease a therapeuticallyeffective amount of an agent toxic to the cells including an analog of aType I ribosome-inactivating protein linked through a cysteine to amolecule which specifically binds to the cell, the analog having thecysteine at an amino acid position corresponding to a position notnaturally available for intermolecular disulfide bonding in the Type Iribosome-inactivating protein and the cysteine being located at aposition in the amino acid sequence of the analog from the positioncorresponding to position 251 in SEQ ID NO: 1 to the carboxyl terminalposition of the analog.

The present invention also provides an analog of a Type Iribosome-inactivating protein, wherein the analog has a cysteineavailable for intermolecular disulfide bonding located at an amino acidposition corresponding to a position not naturally available forintermolecular disulfide bonding in the Type I ribosome-inactivatingprotein and corresponding to a position on the surface of ricin A-chainin its natural conformation, and wherein the analog retainsribosome-inactivating activity of the Type I ribosome-inactivatingprotein.

Such an analog may be an analog wherein the Type I ribosome inactivatingprotein is gelonin, and is preferably an analog of gelonin wherein thecysteine is at position 10 of the amino acid sequence of the analog asencoded in a vector in a host cell deposited as A.T.C.C. Accession No.69008. Other such gelonin analogs include those wherein the cysteine isat a position 60 in the amino acid sequence of the gelonin analog.

The present invention further provides an analog of a Type Iribosome-inactivating protein wherein the analog includes only a singlecysteine. Such an analog may be an analog of gelonin and is preferablyan analog wherein the single cysteine is at position 10, position 44,position 50 or position 247 in the amino acid sequence of the analog,but the cysteine may be located at other positions defined by theinvention as well.

The present invention provides a polynucleotide encoding an analog of aType I ribosome-inactivating protein, wherein the analog has a cysteineavailable for intermolecular disulfide bonding located at an amino acidposition corresponding to a position not naturally available forintermolecular disulfide bonding in the Type I ribosome-inactivatingprotein and corresponding to a position on the surface of ricin A-chainin its natural conformation, and wherein the analog retainsribosome-inactivating activity of the Type I ribosome-inactivatingprotein.

According to the present invention, a method for preparing an analog ofa Type I ribosome-inactivating protein may include the step ofexpressing in suitable host cell a polynucleotide encoding a Type Iribosome-inactivating protein having a cysteine available forintermolecular disulfide bonding substituted at an amino acid positioncorresponding to a position not naturally available for disulfidebonding in the Type I ribosome-inactivating protein, the cysteine islocated at a position corresponding to an amino acid position on thesurface of ricin A-chain in its natural conformation and which analogretains ribosome-inactivating activity of the Type Iribosome-inactivating protein.

The present invention provides an agent toxic to a cell including ananalog of a Type I ribosome-inactivating protein linked by a disulfidebond through a cysteine to a molecule which specifically binds to thecell, wherein the analog has a cysteine available for intermoleculardisulfide bonding located at an amino acid position corresponding to aposition not naturally available for intermolecular disulfide bonding inthe Type I ribosome-inactivating protein and corresponding to a positionon the surface of ricin A-chain in its natural conformation, and whereinthe analog retains ribosome-inactivating activity of the Type Iribosome-inactivating protein.

A method according to the present invention for preparing an agent toxicto a cell may include the step of linking an analog of a Type Iribosome-inactivating protein through a cysteine to a molecule whichspecifically binds to the cell, which analog has a cysteine availablefor intermolecular disulfide bonding located at an amino acid positioncorresponding to a position not naturally available for intermoleculardisulfide bonding in the Type I ribosome-inactivating protein andcorresponding to a position on the surface of ricin A-chain in itsnatural conformation, and which analog retains ribosome-inactivatingactivity of the Type I ribosome-inactivating protein.

A method according to the present invention for treating a disease inwhich elimination of particular cells is a goal includes the step ofadministering to a patient having the disease a therapeuticallyeffective amount of an agent toxic to the cells wherein the agentincludes an analog of a Type I ribosome-inactivating protein linked by adisulfide bond through a cysteine to a molecule which specifically bindsto the cell, which analog has a cysteine available for intermoleculardisulfide bonding located at an amino acid position corresponding to aposition not naturally available for intermolecular disulfide bonding inthe Type I ribosome-inactivating protein and corresponding to a positionon the surface of ricin A-chain in its natural conformation, and whichanalog retains ribosome-inactivating activity of the Type Iribosome-inactivating protein.

The present invention also provides a purified and isolatedpolynucleotide encoding natural sequence gelonin, and a host cellincluding a vector encoding gelonin of the type deposited as A.T.C.C.Accession No. 68721.

The present invention further provides a purified and isolatedpolynucleotide encoding natural sequence barley ribosome-inactivatingprotein, and a purified and isolated polynucleotide encoding momordinII.

The RIP analogs are particularly suited for use as components ofcytotoxic therapeutic agents and, more specifically, as components ofimmunotoxins. Cytotoxic agents according to the present invention may beused in vivo to selectively eliminate any cell type to which the RIPcomponent is targeted by the specific binding capacity of the secondcomponent. To form an immunotoxin, conjugation to monoclonal antibodies,including chimeric and CDR-grafted antibodies, and antibodydomains/fragments (e.g., Fab, Fab', F(ab')₂, single chain antibodies,and Fv or single variable domains) as well as conjugation to monoclonalantibodies genetically engineered to include free cysteine residues arewithin the scope of the present invention. Examples of Fab' and F(ab')₂fragments useful in the present invention are described in copending,co-owned U.S. patent application Ser. No. 07/714,175,filed June 14,1991, now abandoned which is incorporated by reference herein. RIPsaccording to the present invention may also be conjugated to targetingagents other than antibodies, for example, lectins which bind to cellshaving particular surface carbohydrates or hormones which bindspecifically to cells having particular receptors.

Cytotoxic agents according to the present invention are suited fortreatment of diseases where the elimination of a particular cell type isa goal, such as autoimmune disease, cancer and graft-versus-hostdisease. The cytotoxic agents are also suited for use in causingimmunosuppression and in treatment of infections by viruses such as theHuman Immunodeficiency Virus.

Specifically illustrating polynucleotide sequences according to thepresent invention are the inserts in the plasmid pING3731 in E. coliMC1061 (designated strain G274) and in the plasmid pING3803 in E. coliE104 (designated strain G275), both deposited with the American TypeCulture Collection (A.T.C.C.), Rockville, Md., on Oct. 2, 1991, andassigned A.T.C.C. Accession Nos. 68721 and 68722, respectively.Additional polynucleotide sequences illustrating the invention are theinserts in the plasmid pING3746 in E. coli E104 (designated strain G277)and in the plasmid pING3737 in E. coli E104 (designated strain G276),which were both deposited with the A.T.C.C. on Jun. 9, 1992, and wererespectively assigned Accession Nos. 69008 and 69009.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a computer-generated alignment of the amino acid sequence ofthe ricin A-chain (RTA) (SEQ ID NO: 1) with the amino acid sequence ofthe Type I ribosome-inactivating protein gelonin (SEQ ID NO: 2), whereinstarred positions indicate amino acids invariant among the ricin A-chainand the Type I RIPs;

FIG. 2 is a computer-generated alignment of the amino acid sequence ofthe ricin A-chain (SEQ ID NO: 1) with the amino acid sequence of theType I ribosome-inactivating protein BRIP (SEQ ID NO: 3), whereinstarred positions indicate amino acids invariant among the ricin A-chainand the Type I RIPs;

FIG. 3 is a computer-generated alignment of the amino acid sequence ofthe ricin A-chain (SEQ ID NO: 1) with the amino acid sequence of theType I ribosome-inactivating protein momordin II (MOMOII) (SEQ ID NO:4), wherein starred positions indicate amino acids invariant among thericin A-chain and the Type I RIPs;

FIG. 4 is a computer-generated alignment of the amino acid sequence ofthe ricin A-chain (SEQ ID NO: 1) with the amino acid sequence of theType I ribosome-inactivating protein luffin (SEQ ID NO: 5), whereinstarred positions indicate amino acids invariant among the ricin A-chainand the Type I RIPs;

FIG. 5 is a computer,generated alignment of the amino acid sequence ofthe ricin A-chain (SEQ ID NO: 1) with the amino acid sequence of theType I ribosome-inactivating protein αtrichosanthin (TRICHO) (SEQ ID NO:6), wherein starred positions indicate amino acids invariant among thericin A-chain and the Type I RIPs;

FIG. 6 is a computer-generated alignment of the amino acid sequence ofthe ricin A-chain (SEQ ID NO: 1) with the amino acid sequence of theType I ribosome-inactivating protein momordin I (MOMOI) (SEQ ID NO: 7),wherein starred positions indicate amino acids invariant among the ricinA-chain and the Type I RIPs;

FIG. 7 is a computer-generated alignment of the amino acid sequence ofthe ricin A-chain (SEQ ID NO: 1) with the amino acid sequence of theType I ribosome-inactivating protein Mirabilis anti-viral protein (MAP)(SEQ ID NO: 8), wherein starred positions indicate amino acids invariantamong the ricin A-chain and the Type I RIPs;

FIG. 8 is a computer-generated alignment of the amino acid sequence ofthe ricin A-chain (SEQ ID NO: 1) with the amino acid sequence of theType I ribosome-inactivating protein pokeweed antiviral protein fromseeds (PAPS) (SEQ ID NO: 9), wherein starred positions indicate aminoacids invariant among the ricin A-chain and the Type I RIPs;

FIG. 9 is a computer-generated alignment of the amino acid sequence ofthe ricin A-chain (SEQ ID NO: 1) with the amino acid sequence of theType I ribosome-inactivating protein saporin 6 (SAP6) (SEQ ID NO: 10),wherein starred positions indicate amino acids invariant among the ricinA-chain and the Type I RIPs;

FIG. 10 sets out the DNA and deduced amino acid sequences of the Type IRIP gelonin (SEQ ID NOs: 11 and 2, respectively) as well as indicatingthe correspondence of oligonucleotide primers utilized for cloning andexpression of the gene to the sequences;

FIG. 11 sets out the DNA and deduced amino acid sequences of the Type IRIP BRIP (SEQ ID NOs: 12 and 3, respectively) as well as indicating thecorrespondence of oligonucleotide primers utilized for cloning andexpression of the gene and construction of analogs of the gene to thesequences; and

FIG. 12 sets out the DNA and deduced amino acid sequences of the Type IRIP momordin II (SEQ ID NOs: 13 and 4, respectively) as well asindicating the correspondence of oligonucleotide primers utilized forcloning and expression of the gene to the sequences.

DETAILED DESCRIPTION

Nucleotide sequences of genes encoding three plant Type I RIPs andexpression vectors containing the genes are provided by the presentinvention. A first plant RIP, gelonin, is produced by seeds of Geloniummultiflorum, a plant of the Euphorbiaceae family native to the tropicalforests of eastern Asia, while a second plant RIP, BRIP, is synthesizedby the common cereal grain barley. Momordin II, a third plant RIP, isproduced in Momordica balsamina seeds. Analogs of BRIP are also providedby the present invention. The analogs were genetically engineered toinclude a cysteine free to participate in a intermolecular disulfidebond and were conjugated to antibody molecules without non-specificchemical derivatization of the RIP with crosslinking agents.

Type I RIP analogs of the present invention offer distinct advantagesover the natural proteins for use as components of immunotoxins.Chemical treatment to introduce free sulfhydryl groups in the naturalproteins lacking free cysteines typically involves the non-selectivemodification of amino acid side chains. This non-selectivity oftenresults in antibodies conjugated to different sites on different RIPmolecules (i.e., a heterogeneous population of conjugates) and also in adecrease in RIP activity if antibodies are conjugated in or nearimportant regions of the RIP (e.g., the active site or regions involvedin translocation across cell membranes). In contrast, RIP analogsaccording to the present invention can be conjugated to a singleantibody through a disulfide bond to a specific residue of the analogresulting in reduced batch to batch variation of the immunoconjugatesand, in some cases, immunoconjugates with enhanced properties (e.g.,greater cytotoxicity or solubility).

Type I plant RIPs, as well as bacterial RIPs such as shiga andshiga-like toxin A-chains, are homologous to the ricin A-chain and areuseful in the present invention.

Type I RIPs may be defined and sites for substitution of a cysteine in aRIP may be identified by comparing the primary amino acid sequence ofthe RIP to the natural ricin A-chain amino acid sequence, the tertiarystructure of which has been described in Katzin et al., Proteins, 10,251-259 (1991), which is incorporated by reference herein.

Amino acid sequence alignment defines Type I RIPs in that the ricinA-chain and the Type I plant RIPs have nine invariant amino acids incommon. Based on the ricin sequence the invariant amino acids aretyrosine₂₁, arginine₂₉, tyrosines₈₀, tyrosine₁₂₃, leucine₁₄₄, glutamicacids₁₇₇, alanine₁₇₈, arginine₁₈₀, and tryptophan₂₁₁. The ricin A-chainmay be used as a model for the three-dimensional structure of Type IRIPs. A protein lacking a cysteine available for conjugation whilehaving ribosome-inactivating activity and having the nine invariantamino acids when its primary sequence is compared to the primarysequence of the ricin A-chain [according to the alignment algorithm ofMyers et al., CABIOS COMMUNICATIONS, 4(1), 11-17 (1988), implemented bythe PC/GENE program PALIGN (Intelligenetics, Inc., Mountain View,Calif.) and utilizing the Dayhoff Mutation Data Matrix (MDM-78) asdescribed in Schwartz et al., pp. 353-358 in Atlas of Protein Sequenceand Structure, 5 Supp. 3, National Biomedical Research Foundation,Washington, D.C. (1978)] is defined as a Type I RIP herein and isexpected to be useful in the present invention. "Corresponding" refersherein to amino acid positions that align when two amino acid sequencesare compared by the strategy of Myers et al., supra.

The primary amino acid sequences of the Type I RIPs gelonin, BRIP,momordin II, luffin [see Islam et al., Agricultural Biological Chem.,54(5), 1343-1345 (199)], αtrichosanthin [see Chow et al., J. Biol.Chem., 265, 8670-8674 (1990)], momordin I [see Ho et al., BBA, 1088,311-314 (1991)], Mirabilis anti-viral protein [see Habuka et al., J.Biol. Chem., 264(12), 6629-6637 (1989)], pokeweed antiviral proteinisolated from seeds [see Kung et al., Agric. Biol. Chem., 54(12),3301-3318 (1990)] and saporin [see Benatti et al., Eur. J. Biochem.,183,465-470 (1989)] are individually aligned with the primary sequenceof the ricin A-chain [see Halling et al., Nucleic Acid Res., 13,8019-8033 (1985)] in FIGS. 1-9, respectively, according to the algorithmof Myers et al., supra, as specified above.

FIGS. 1-9 may be utilized to predict the amino acid positions of theType I RIPs where cysteine residues may be substituted. Preferred aminoacids for cysteine substitution are on the surface of the molecule andinclude any solvent accessible amino acids that will not interfere withproper folding of the protein if replaced with a cysteine. A region ofthe ricin A-chain comprising such amino acids is the carboxyl terminalregion. Amino acids that should be avoided for replacement are thosecritical for proper protein folding, such as proline, and those that aresolvent inaccessible. Also to be avoided are the nine amino acidsinvariant among RIPs, and the amino acids in or near regions comprisingthe active site of ricin A-chain as depicted in FIG. 6 of Katzin et al.,supra.

Therefore, a preferred region of substitution for Type I RIPs is theircarboxyl terminal region which is solvent accessible and corresponds tothe carboxyl terminal region where Type II RIP A-chains and B-chains arenaturally linked by a disulfide bond. As shown in the examples, acysteine may be substituted in positions in the amino acid sequence of aType I RIP from the position corresponding to position 251 in SEQ ID NO:1 to the carboxyl terminal position of said Type I RIP, resulting in RIPanalogs which retain enzymatic activity and gain disulfide cross-linkingcapability. One preferred cysteine substitution position is near theposition which corresponds to the cysteine at position 259 in the ricinA-chain.

Immunotoxins specifically illustrating the present invention areparticularly suited for use in treatment of human autoimmune diseasewhere T-cell function is implicated. Treatment of autoimmune diseaseswith immunotoxins is described in co-owned U.S. patent application Ser.No. 306,433 filed on Sep. 13, 1991, now abandoned which is incorporatedby reference herein. Examples of autoimmune diseases are systemic lupuserythematosus, scleroderma diseases (including lichen sclerosus, morpheaand lichen planus), rheumatoid arthritis, chronic thyroiditis, pemphigusvulgaris, diabetes mellitus type 1, progressive systemic sclerosis,aplastic anemia, myasthenia gravis, myositis, Sjogrens disease, Crohn'sdisease, ulcerative colitis, and primary biliary cirrhosis. Autoimmunityis also implicated in multiple sclerosis, uveitis, psoriasis andMeniere's disease. A general description of various autoimmune diseasesmay be found in Rose and Mackey, Eds., The Autoimmune Diseases, AcademicPress (1985).

The immunotoxins may be administered to a patient either singly or in acocktail containing two or more immunotoxins, other therapeutic agents,compositions, or the like, including, but not limited to,immunosuppressive agents, tolerence-inducing agents, potentiators andside-effect relieving agents. Particularly preferred areimmunosuppressive agents useful in suppressing allergic reactions of ahost. Preferred immunosuppressive agents include prednisone,prednisolone, DECADRON (Merck, Sharp & Dohme, West Point, Pa.),cyclophosphamide, cyclosporine, 6-mercaptopurine, methotrexate,azathioprine and i.v. gamma globulin or their combination. Preferredpotentiators include monensin, ammonium chloride, perhexiline,verapamil, amantadine and chloroquine. All of these agents areadministered in generally-accepted efficacious dose ranges such as thosedisclosed in the Physician's Desk Reference, 41st Ed., Publisher EdwardR. Barnhart, N.J. (1987). Patent Cooperation Treaty (PCT) patentapplication WO 89/069767 published on Aug. 10, 1989, disclosesadministration of an immunotoxin as an immunosuppressive agent and isincorporated by reference herein.

Anti-T cell immunotoxins may be formulated into either an injectable ortopical preparation. Parenteral formulations are known and are suitablefor use in the invention, preferably for intramuscular or intravenousadministration. The formulations containing therapeutically-effectiveamounts of anti-T cell immunotoxins are either sterile liquid solutions,liquid suspensions or lyophilized versions, and optionally containstabilizers or excipients. Lyophilized compositions are reconstitutedwith suitable diluents, e.g., water for injection, saline, 0.3% glycineand the like, at a level of about from 0.01 mg/kg of host body weight to10 mg/kg where the biological activity is less than or equal to 20 ng/mlwhen measured in a reticulocyte lysate assay. Typically, thepharmaceutical compositions containing anti-T cell immunotoxins will beadministered in a therapeutically effective dose in a range of fromabout 0.01 mg/kg to about 5 mg/kg of the patient. A preferred,therapeutically effective dose of the pharmaceutical compositioncontaining anti-T cell immunotoxin will be in a range of from about 0.01mg/kg to about 0.5 mg/kg body weight of the patient administered overseveral days to two weeks by daily intravenous infusion, each given overa one hour period, in a sequential patient dose-escalation regimen.

Anti-T cell immunotoxin is formulated into topical preparations forlocal therapy by including a therapeutically effective concentration ofanti-T cell immunotoxin in a dermatological vehicle. The amount ofanti-T cell immunotoxin to be administered, and the anti-T cellimmunotoxin concentration in the topical formulations, depends upon thevehicle selected, the clinical condition of the patient, the systemictoxicity and the stability of the anti-T cell immunotoxin in theformulation. Thus, a physician knows to employ the appropriatepreparation containing the appropriate concentration of anti-T cellimmunotoxin in the formulation, as well as the appropriate amount offormulation to administer depending upon clinical experience with thepatient in question or with similar patents. The concentration of anti-Tcell immunotoxin for topical formulations is in the range of greaterthan from about 0.1 mg/ml to about 25 mg/ml. Typically, theconcentration of anti-T cell immunotoxin for topical formulations is inthe range of greater than from about 1 mg/ml to about 20 mg/ml. Soliddispersions of anti-T cell immunotoxin as well as solubilizedpreparations can be used. Thus, the precise concentration to be used inthe vehicle is subject to modest experimental manipulation in order tooptimize the therapeutic response. Greater than about 10 mg anti-T cellimmunotoxin/100 grams of vehicle may be useful with 1% w/w hydrogelvehicles in the treatment of skin inflammation. Suitable vehicles, inaddition to gels, are oil-in-water or water-in-oil emulsions usingmineral oils, petroleum and the like.

Anti-T cell immunotoxin is optionally administered topically by the useof a transdermal therapeutic system [Barry, Dermatological Formulations,p. 181 (1983) and literature cited therein]. While such topical deliverysystems have been designed for transdermal administration of lowmolecular weight drugs, they are capable of percutaneous delivery. Theymay be readily adapted to administration of anti-T cell immunotoxin orderivatives thereof and associated therapeutic proteins by appropriateselection of the rate-controlling microporous membrane.

Topical preparations of anti-T cell immunotoxin either for systemic orlocal delivery may be employed and may contain excipients as describedabove for parenteral administration and other excipients used in atopical preparation such as cosolvents, surfactants, oils, humectants,emollients, preservatives, stabilizers and antioxidants. Anypharmacologically-acceptable buffer may be used, e.g., Tris or phosphatebuffers. The topical formulations may also optionally include one ormore agents variously termed enhancers, surfactants, accelerants,adsorption promoters or penetration enhancers, such as an agent forenhancing percutaneous penetration of the anti-T cell immunotoxin orother agents. Such agents should desirably possess some or all of thefollowing features as would be known to the ordinarily skilled artisan:pharmacological inertness, non-promotive of body fluid or electrolyteloss, compatible with anti-T cell immunotoxin (non-inactivating), andcapable of formulation into creams, gels or other topical deliverysystems as desired.

Anti-T cell immunotoxin may also be administered by aerosol to achievelocalized delivery to the lungs. This is accomplished by preparing anaqueous aerosol, liposomal preparation or solid particles containingimmunotoxin. Ordinarily, an aqueous aerosol is made by formulating anaqueous solution or suspension of anti-T cell immunotoxin together withconventional pharmaceutically acceptable carriers and stabilizers. Thecarriers and stabilizers vary depending upon the requirements for theparticular anti-T cell immunotoxin, but typically include: nonionicsurfactants (Tweens, Pluronics, or polyethylene glycol); innocuousproteins like serum albumin, sorbitan esters, oleic acid, lecithin;amino acids such as glycine; and buffers, salts, sugars or sugaralcohols. The formulations may also include mucolytic agents as well asbronchodilating agents. The formulations are sterile. Aerosols generallyare prepared from isotonic solutions. The particles optionally includenormal lung surfactants.

Aerosols may be formed of the particles in aqueous or nonaqueous (e.g.,fluorocarbon propellant) suspension. Such particles include, forexample, intramolecular aggregates of anti-T cell immunotoxin orderivatives thereof or liposomal or microcapsular-entrapped anti-T cellimmunotoxin or derivatives thereof. The aerosols should be free of lungirritants, i.e., substances which cause acute bronchoconstriction,coughing, pulmonary edema or tissue destruction. However, nonirritatingabsorption-enhancing agents are suitable for use herein. Sonicnebulizers are preferably used in preparing aerosols. Sonic nebulizersminimize exposing the anti-T cell immunotoxin or derivatives thereof toshear, which can result in degradation of anti-T cell immunotoxin.

Anti-T cell immunotoxin may be administered systemically, rather thantopically, by injection intramuscularly, subcutaneously, intrathecallyor intraperitoneally or into vascular spaces, particularly into thejoints, e.g., intraarticular injection at a dosage of greater than about1 μg/cc joint fluid/day. The dose will be dependent upon the propertiesof the anti-T cell immunotoxin employed, e.g., its activity andbiological half-life, the concentration of anti-T cell immunotoxin inthe formulation, the site and rate of dosage, the clinical tolerance ofthe patient involved, the autoimmune disease afflicting the patient andthe like, as is well within the skill of the physician.

The anti-T cell immunotoxin of the present invention may be administeredin solution. The pH of the solution should be in the range of pH 5 to9.5, preferably pH 6.5 to 7.5. The anti-T cell immunotoxin orderivatives thereof should be in a solution having a suitablepharmaceutically-acceptable buffer such as phosphate,Tris(hydroxymethyl)aminomethane-HCl or citrate and the like. Bufferconcentrations should be in the range of 1 to 100 mM. The solution ofanti-T cell immunotoxin may also contain a salt, such as sodium chlorideor potassium chloride in a concentration of 50 to 150 mM. An effectiveamount of a stabilizing agent such as an albumin, a globulin, a gelatin,a protamine or a salt of protamine may also be included, and may beadded to a solution containing anti-T cell immunotoxin or to thecomposition from which the solution is prepared.

Systemic administration of anti-T cell immunotoxin is made daily and isgenerally by intramuscular injection, although intravascular infusion isacceptable. Administration may also be intranasal or by othernonparenteral routes. Anti-T cell immunotoxin may also be administeredvia microspheres, liposomes or other microparticulate delivery systemsplaced in certain tissues including blood. Topical preparations areapplied daily directly to the skin or mucosa and are then preferablyoccluded, i.e., protected by overlaying a bandage, polyolefin film orother barrier impermeable to the topical preparation.

The following examples illustrate practice of the invention in thecloning of the genes encoding the Type I RIPs gelonin, BRIP and momordinII, and in the production of analogs of gelonin and BRIP and ofimmunoconjugates of the analogs. The examples are not to be construed aslimiting the invention.

Example 1

The cloning of the gelonin gene according to the present inventionobviates the requirement of purifying the RIP gene product from itsrelatively scarce natural source, G. multiflorum seeds, and allowsdevelopment of gelonin analogs conjugatable to antibodies without priorchemical derivatization. One formidable hurdle in the cloning of thegene was that the available Gelonium seeds are old and inviable makingpreparation of intact messenger RNA from the seeds impossible. Cloningthe gene from cDNA prepared from messenger RNA was thus impractical andtotal RNA was utilized to generate cDNA. Using total RNA to make cDNAunder normal circumstances, i.e., when mRNA may be utilized, is notdesirable because total RNA typically comprises about 95% ribosomal RNA.

Preparation of RNA from G. Multiflorum Seeds

Total RNA was prepared from Gelonium seeds (Dr. Michael Rosenblum, M. D.Anderson Cancer Center, Houston, Tex.) by a modification of theprocedure for preparation of plant RNA described in Ausubel et al.,eds., Current Protocols in Molecular Biology, Wiley & Sons, 1989.Briefly, 4.0 grams of seeds were ground to a fine powder in a pre-cooled(-70° C.) mortar and pestle with liquid N₂. The powder was added to 25ml Grinding buffer (0.18M Tris, 0.09M LiCl, 4.5 mM EDTA, 1% SDS, pH 8.2)along with 8.5 ml of phenol equilibrated with TLE (0.2M Tris, 0.1M LiCl,5 mM EDTA pH 8.2). The mixture was homogenized using a Polytron PT-1035(#5 setting). 8.5 ml of chloroform was added, mixed and incubated at 50°C. for 20 minutes. The mixture was centrifuged at 3K for 20 minutes in arotor precooled to 4° C. and the aqueous phase was transferred to a newtube. 8.5 ml of phenol was added followed by 8.5 ml of chloroform andthe mixture was recentrifuged. This extraction was repeated 3 times. TheRNA in the aqueous phase was then precipitated by adding 1/3 volume 8MLiCl, and incubated at 4° C. for 16 hours. Next, the RNA was pelleted bycentrifugation for 20 minutes at 4° C. The pellet was washed with 5 mlof 2M LiCl, recentrifuged and resuspended in 2 ml of water. The RNA wasprecipitated by addition of NaOAc to 0.3M and 2 volumes of ethanol. TheRNA was stored in 70% ethanol at -70° C.

cDNA Preparation

cDNA was prepared from total Gelonium RNA by two similar methods.

The first method involved making a cDNA library in the bacterialexpression plasmid pcDNAII using the Librarian II cDNA LibraryConstruction System kit (Invitrogen). About 5 μg of total RNA wasconverted to first strand cDNA with a 1:1 mixture of random primers andoligo-dT. Second strand synthesis with DNA polymerase I was performed asdescribed by the system manufacturer. Double stranded cDNA was ligatedto BstX1 linkers and size fractionated. Pieces larger than about 500 bpwere ligated into the expression vector provided in the kit. Individualvectors were introduced into E. coli either by transformation intohigh-efficiency competent cells or by electroporation intoelectrocompetent cells. Electroporation was performed with a BTX100 unit(BTX, San Diego, Calif.) in 0.56μ Flatpack cells as recommended by BTXbased on the method of Dower et al., Nucleic Acids Res., 16, 6127-6145(1988), at a voltage amplitude of 850 V and a pulse length of 5 mS. Theresulting library consisted of approximately 150,000 colonies.

The second method involved generating cDNA using the RNA-PCR kit sold byPerkin-Elmer-Cetuso About 100 ng of total Gelonium RNA was used astemplate for cDNA synthesis.

Determination of the Gelonin Protein Sequence

The partial sequence of the native gelonin protein was determined bydirect amino acid sequence analysis by automated Edman degradation asrecommended by the manufacturer using an Applied Biosystems model 470Aprotein sequencer. Proteolytic peptide fragments of gelonin (isolatedfrom the same batch of seeds as the total RNA) were sequenced.

Cloning of the Gelonin Gene

Three overlapping gelonin cDNA fragments were cloned and a compositegelonin gene was assembled from the three fragments.

(1) Cloning of the Fragment Encoding the Middle Amino Acids of Geloninin Vector pING3823.

Degenerate DNA primers based on the gelonin partial amino acid sequenceswere used to amplify by PCR, segments of the cDNA generated with thePerkin-Elmer-Cetus kit. Six primers were designed based on regions ofthe gelonin amino acid sequence where degeneracy of the primers could beminimized. Appropriate pairs of primers were tested for amplification ofa gelonin gene fragment. When products of the expected DNA size wereidentified as ethidium bromide-stained DNA bands on agarose gels, theDNA was treated with T4 DNA polymerase and then purified from an agarosegel. Only the primer pair consisting of primers designated gelo-7 andgelo-5 yielded a relatively pure product of the expected size. Thesequences of degenerate primers gelo-7 and gelo-5 are set out belowusing IUPAC nucleotide symbols. ##STR1## Primer gelo-7 corresponds toamino acids 87-97 of gelonin while primer gelo-5 corresponds to aminoacids 226-236. The blunt-ended DNA fragment (corresponding to aminoacids 87 to 236 of gelonin) generated with primers gelo-7 and gelo-5 wascloned into pUC18 (BRL, Gaithersburg, Md.). The DNA sequence of theinsert was determined, and the deduced amino acid sequence based on theresulting DNA sequence matched the experimentally determined geloninamino acid sequence. The clone containing this gelonin segment isdenoted pING3726.

The insert of clone pING3726 was labeled with ³² p and used as a probeto screen the 150,000-member Gelonium cDNA library. Only one clonehybridized to the library plated in duplicate. This clone was purifiedfrom the library and its DNA sequence was determined. The clone containsa fragment encoding 185 of the 270 amino acids of gelonin (residues25-209) and is denoted pING3823.

(2) Cloning of the Fragment Encoding the N-terminal Amino Acids ofGelonin.

Based on the sequence determined for the gelonin gene segment inpING3726, exact oligonucleotide primers were designed as PCRamplification primers to be used in conjunction with a degenerate primerto amplify a 5' gelonin gene fragment and with a nonspecific primer toamplify a 3' gelonin gene fragment. cDNA generated using thePerkin-Elmer-Cetus RNA-PCR kit was amplified.

To amplify the 5'-end of the gelonin gene, PCR amplification with adegenerate primer gelo-1 and an exact primer gelo-10 was performed. Thesequences of the primers are set out below. ##STR2## Primer gelo-1corresponds to amino acids 1-11 of the gelonin gene while primer gelo-10corresponds to amino acids 126-133. The product from the reaction wasre-amplified with gelo-1 (SEQ ID NO: 16) and gelo-11 (an exact primercomprising sequences encoding amino acids 119-125 of gelonin) to conferspecificity to the reaction product. The sequence of primer gelo-11 islisted below. ##STR3## Hybridization with an internal probe confirmedthat the desired specific gelonin DNA fragment was amplified. Thisfragment was cloned into pUC18, and the vector generated was designatedpING3727. The fragment was sequenced revealing that the region of thefragment (the first 27 nucleotides) corresponding to part of thedegenerate primer gelo-1 could not be translated to yield the amino acidsequence upon which primer gelo-1 was originally based. This was notunexpected considering the degeneracy of the primer. The fragment wasreamplified from the Gelonium cDNA with exact primers gelo-11 (SEQ IDNO: 18) and gelo-5' (which extends upstream of the 5' end of the geloningene in addition to encoding the first 16 amino acids of gelonin). Thesequence of primer gelo-5' is set out below. ##STR4## The resulting DNAfragment encodes the first 125 amino acids of gelonin. While themajority of the sequence is identical to the natural gelonin gene, thefirst 32 nucleotides of the DNA fragment may not be. For the purposes ofthis application this N-terminal fragment is referred to as fragmentGELl-125.

(3) Cloning of the Fragment Encoding the C-terminal Amino Acids ofGelonin.

To amplify the 3'-end of the gelonin gene as well as 3' untranslatedsequences, PCR amplification with exact primers gelo-9 and XE-dT wasperformed. The sequence of each of the primers is set out below.##STR5## Primer gelo-9 corresponds to amino acids 107-113 of gelonin.Primer XE-dT consists of an 3' oligo-dT portion and a 5' portioncontaining the restriction sites HindIII and XhoI, and will prime anypoly A-containing cDNA. The reaction product was reamplified with exactprimers gelo-8 and XE. The sequences of primers gelo-8 and XE are setout below. ##STR6## Primer gelo-8 consists of sequences encoding aminoacids 115-120 of gelonin while the primer XE corresponds to the 5'portion of the XE-dT primer which contains HindIII and XhoI restrictionsites. Hybridization with internal probes confirmed that the desiredgelonin gene fragment was amplified. The fragment was cloned into pUC18by two different methods. First, it was cloned as a blunt-ended fragmentinto the SmaI site of pUC18 (the resulting vector was designatedpING3728) and, second, it was cloned as an EcoRI to HindIII fragmentinto pUC18 (this vector was designated pING3729). Both vector insertswere sequenced. The insert of pING3728 encodes amino acids 114-270 ofgelonin, while the insert of pING3729 encodes amino acids 184-270 ofgelonin plus other 3' sequences.

(4) Assembly of the overlapping gelonin DNA fragments into a compositegelonin gene

To reassemble the C-terminal two-thirds of the gelonin gene, vectorpING3729 was cut with SspI (one SspI site is located within the vectorand the second is located about 80 bp downstream of the terminationcodon of the insert in the vector) and an XhoI linker (8 bp, New EnglandBiolabs) was ligated to the resulting free ends. The DNA was then cutwith XhoI and EcoRI, and the 350 bp fragment generated, encoding aminoacids 185-270 of gelonin, was isolated. This 350 bp fragment was ligatedadjacent to a NcoI to EcoRI fragment from pING3823 encoding amino acids37-185 of gelonin in a intermediate vector denoted pING3730, thusreassembling the terminal 87% of the gelonin gene (amino acids 37-270).

Next, fragment GEL1-125 was cut with SmaI and NcoI, resulting in afragment encoding amino acids 1-36 of gelonin which was ligated alongwith the NcoI to XhoI fragment of pING3730 into the vector pIC100.[pIC100 is identical to pING1500 described in Better et al., Science,240, 1041-1043 (1988), except that it lacks 37 bp upstream of the pelBleader sequence. The 37 bp were eliminated by digestion of pING1500 withSphI and EcoRI, treatment with T4 polymerase and religation of thevector. This manipulation regenerated an EcoRI site in the vector whileeliminating other undesirable restriction sites.] Before ligation, thevector pIC100 had previously been digested with SstI, treated with T4polymerase, and cut with XhoI. The ligation generated a new vectorcontaining a complete gelonin gene that was designated plasmid pING3731(A.T.C.C. Accession No. 68721). The complete DNA sequence of the geloningene is set out in SEQ ID NO: 11 and in FIG. 10.

Construction of Expression Vectors Containing the Gelonin Gene

A first E. coli expression vector was constructed containing the geloningene linked to the Erwinia carotovora pelB leader sequence, and to theSalmonella typhimurium araB promoter. A basic vector containing the araBpromoter is described in co-owned U.S. Pat. No. 5,028,530 issued Jul. 2,1991 which is incorporated herein by reference. The vector containingthe araB promoter was cut with EcoRI and XhoI. Two DNA fragments werethen ligated in tandem immediately downstream of the promoter. Thefragment ligated adjacent to the promoter was a 131 bp fragment derivedfrom SstI digestion, T4 polymerase treatment and digestion with EcoRI ofthe pIC100 vector which includes the leader sequence of the E.carotovora pelB gene. The translated leader sequence is a signal forsecretion of the respective protein through the cytoplasmic membrane.The fragment ligated downstream of the leader sequence was a SmaI toXhoI fragment from pING3731 which contains the complete gelonin gene.Thus, the expression vector contains the gelonin gene linked to the pelBleader sequence and the arab promoter. This plasmid is designatedpING3733.

A second expression vector may be constructed that is identical to thefirst except that the gelonin gene sequences encoding the nineteenC-terminal amino acids of gelonin are not included. The cDNA sequence ofthe gelonin gene predicted a 19 residue C-terminal segment that was notdetected in any peptide fragments generated for determination of thegelonin amino acid sequence. These 19 amino acids may represent apeptide segment that is cleaved from the mature toxinpost-translationally, i.e. that is not present in the native protein. Asimilar C-terminal amino acid segment was identified in the plant toxinαtrichosanthin [Chow et al., J. Biol. Chem., 265, 8670-8674 (1990)].Therefore, the expression product without the C-terminal fragment may beof importance.

For construction of a gelonin expression vector without the 19C-terminal amino acids of gelonin, PCR was used to amplify and alter the3'-end of the gene. pING3728 was amplified with primers gelo-14 andgelo-9 (SEQ ID NO: 20). The sequence of primer gelo-14 is set out below.##STR7## Primer gelo-14, which corresponds to gelonin amino acids245-256, introduces a termination codon (underlined in the primersequence) in the gelonin gene sequence which stops transcription of thegene before the sequences encoding the terminal 19 amino acids of thegelonin and also introduces a XhoI site immediately downstream of thetermination codon. The PCR product was cut with XhoI and EcoRI, and theresulting 208 bp fragment encoding amino acids 185-251 of gelonin waspurified from an agarose gel. This fragment was ligated adjacent to theNcoI to EcoRI fragment from pING3823 encoding amino acids 37-185 ofgelonin to generate plasmid pING3732. A final expression vector,pING3734, containing a gelonin gene with an altered 3'-end was generatedby substituting an NcoI to XhoI fragment encoding amino acids 37-251 ofgelonin from pING3732 into pING3733.

Identification of the Native Gelonin 5'-End

Inverse PCR was used to identify a cDNA clone encoding the 5'-end of themature gelonin gene. 5 μg of total G. multiflorum RNA was converted tocDNA using the Superscript Plamid System (BRL, Gaithersburg, Md.) withGelo-11 (SEQ ID NO: 18) as a primer. Gelonin cDNA was self-ligated togenerate covalent circular DNA and the ligated DNA was amplified by PCRwith oligonucleotides Gelo-9 (SEQ ID NO: 20) and Gelo-16. The sequenceof primer Gelo-16 is set out below. ##STR8## The PCR product wassize-fractionated on an agarose gel and DNAs larger than 300 bp werecloned into SmaI cut pUC18. Several clones were sequenced with theprimer Gelo-18, the sequence of which is set out below. ##STR9## A cloneidentified as having the largest gelonin-specific insert was designatedpING3826. The DNA sequence of pING3826 included the first 32 nucleotidesof the natural, mature gelonin gene not necessarily present in geloninexpression plasmids pING3733 and pING3734. The complete DNA sequence ofthe natural gelonin gene is set out in SEQ ID NO: 57.

Construction of Expression Vectors Containing a Gelonin Gene with aNatural 5' End

Derivatives of expression vectors pING3733 and pING3734 (describedabove) containing a gelonin gene with the natural 5' sequence weregenerated as follows. The 5'-end of gelonin was amplified from pING3826with the PCR primers Gelo-16 (SEQ ID NO: 24) and Gelo-17, the sequenceof which is set out below. ##STR10## The 285 bp PCR product was treatedwith T4 polymerase and cut with NcoI. The resulting 100 bp 5'-end DNAfragment was isolated from an agarose gel and ligated adjacent to the120 bp pelB leader fragment from plC100 (cut with SstI, treated with T4polymerase and cut with PstI) into either pING3733 or pING3734 digestedwith PstI and NcoI. The resulting plasmids pING3824 and pING3825 containthe entire native gelonin gene and the native gelonin gene minus thenineteen amino acid carboxyl extension, respectively, linked to the pelBleader and under the transcriptional control of the araB promoter. Thegene construct without the nineteen amino acid carboxyl extension inboth pING3734 and pING3825 encodes a protein product referred to in thisapplication as "recombinant gelonin."

Assembly of Gelonin Genes with Cysteine Residues Available forConjugation

The gelonin protein has two cysteine residues at positions 44 and 50which are linked by an endogenous disulfide bond. The protein containsno free cysteine residue directly available for conjugation toantibodies or other proteins. Analogs of gelonin which contain a freecysteine residue available for conjugation were generated by twodifferent approaches. In one approach, various residues along theprimary sequence of the gelonin were replaced with a cysteine residue,creating a series of analogs which contain an odd number of cysteineresidues. In another approach, one of the two endogenous cysteines wasreplaced by alanine, creating a molecule which lacks an intrachaindisulfide bond but contains a single, unpaired cysteine.

Eight analogs of gelonin were constructed. Six non-cysteine residues ofgelonin were targeted for substitution with a cysteine residue.Comparison of the amino acid sequence of gelonin to the natural aminoacid sequence and tertiary structure of the ricin A-chain (see FIG. 1)suggested that these positions would be at the surface of the moleculeand available for conjugation. Each of the six gelonin analogs include acysteine substituted in place of one of the following residues:lysine₁₀, asparagine₆₀, asparagine₂₃₉, lysine₂₄₄, aspartate₂₄₇, andlysine₂₄₈, and the analogs have respectively been designated Gel_(C10),Gel_(C60), Gel_(C230), Gel_(C244), Gel_(C247), and Gel_(C248),

An analog of gelonin was constructed in which one of the native gelonincysteines that participates in an endogenous disulfide bond was replacedwith a non-cysteine residue. Specifically, the cysteine at position 50was replaced with an alanine residue, creating a gelonin analog(designated Gel_(C44)) which has a cysteine available for disulfidebonding at position 44. The combined series of the foregoing sevenanalogs thus spans the entire length of the mature gelonin protein.

An eighth gelonin analog was constructed in which both native gelonincysteines were replaced with alanines. A ninth analog may be constructedthat has alanine residues substituted in place of both native cysteinesand has a cysteine residue substituted in place of the native aspartateat position 247.

The eight variants of recombinant gelonin were constructed by overlapextension PCR of plasmids pING3734 or pING3825 with syntheticoligonucleotides. The sequences of the primers used for PCR are set outbelow. In each mutagenic primer sequence, the nucleotides encoding thechange to a cysteine or an alanine residue are underlined. ##STR11##

(1) Specifically, a cysteine was introduced at amino acid 247 of gelonin(an aspartic acid which corresponds to the cysteine at position 259 inthe ricin A-chain) by PCR with mutagenic primers GeloC-3-2 and GeloC-4in conjunction with primers HindIII-2 (a primer located in the vectorportion of pING3734 or pING3825), Gelo-9 and Gelo-8. Template DNA(pING3734) was amplified with GeloC-3-2 and HindIII-2 and in aconcurrent reaction with GeloC-4 and Gelo-9. The products of thesereactions were mixed and amplified with the outside primers Gelo-8 andHindIII-2. The reaction product was cut with EcoRI and XhoI, purified,and was inserted into plasmid pING3825 in a three-piece ligation. TheDNA sequence of the Gel_(C247) variant was then verified. The plasmidcontaining the sequence encoding Gel_(C247) was designated pING3737(A.T.C.C. Accession No. 69009).

(2-3) In the same manner, a cysteine residue was introduced in place ofthe amino acid acid at position 248 (a lysine) of gelonin with themutagenic oligonucleotides GeloC-1 and GeloC-2 to generate analogGel_(C248) in plasmid pING3741, and a cysteine residue was introduced atamino acid position 239 (a lysine) with primers GeloC-9 and GeloC-10 togenerate analog Gel_(C239) in plasmid pING3744.

(4) Also in the same manner, a cysteine residue was introduced at aminoacid 244 (a lysine) of gelonin with mutagenic primers GeloC-5 andGeloC-6 to generate analog Gel_(C244) in the plasmid designatedpING3736. This variant was prepared by PCR using plasmid pING3734 astemplate DNA rather than pING3825. It therefore encodes the sameN-terminal gelonin amino acid sequence as plasmids pING3737, pING3741,and pING3744, but includes the PCR primer-derived 5'-end 32 nucleotidesinstead of the native gelonin 5'-end nucleotides.

(5) A cysteine residue was introduced in place of the amino acid (alysine) at position 10 of gelonin by a similar procedure. A cysteine wasintroduced with mutagenic primers GeloC-13 and GeloC-14 by amplifyingpING3825 with araB2 (a vector primer) and GeloC-14, and in a separatereaction, with GeloC-13 and Gelo-11. These reaction products were mixedand amplified with the outside primers araB2 and Gelo-11. The PCRproduct was cut with PstI and NcoI, purified, and cloned back intopING3825 to generate analog Gel₁₀ in the plasmid designated pING3746(A.T.C.C. Accession No. 69008).

(6) The asparagine at position 60 of gelonin 10 was replaced with acysteine residue using two mutagenic oligos, GeloC-15 and GeloC-16, inconjunction with oligos araB2 and Gelo-11 in the same manner as for theGel_(C10) variant. The plasmid encoding the Gel_(C60) analog wasdesignated pING3749.

(7) Another gelonin variant with a free cysteine residue was generatedby replacing one of the two naturally occurring gelonin cysteineresidues, the cysteine a position 50, with an alanine. Plasmid pING3824was amplified with primers GeloC-17 and Gelo-11, and concurrently in aseparate reaction with primers GeloC-19 and araB2. The reaction productswere mixed and amplified with araB2 and Gelo-11. This product was cutwith NcoI and BglII, and cloned back into the vector portion of pING3825to generate pING3747. This analog was designated Gel₄₄ because itcontains a cysteine available for disulfide bonding at amino acidposition 44.

(8) A gelonin variant in which both the cysteine at position 44 and thecysteine at position 55 of gelonin were changed to alanine residues wasconstructed by overlap PCR using the mutagenic oligos GeloC-17 andGeloC-18 in conjunction with primers araB2 and Gelo-11. This analog likethe native gelonin protein has no cysteine residues available forconjugation. The plasmid encoding the analog was designated pING3750.

(9) The ninth gelonin variant, including alanine residues at positions44 and 50 and a cysteine residue at position 247, may be generated fromplasmids pING3824, pING3750 and pING3737. Plasmid pING3824 is digestedwith NcoI and XhoI and the vector fragment is isolated. Plasmid pING3750is cut with NcoI and EcoRI and the 449 bp fragment encoding the portionof gelonin having alanines substituted in the place of cysteines 44 and50 is isolated. Plasmid pING3737 is cut with EcoRI and XhoI and the 210bp fragment encoding the portion of gelonin having a cysteine atposition 247 is isolated. The three fragments are ligated to generate agelonin clone including the three amino acid changes.

Each of the eight gelonin variants constructed was transformed into E.coli strain E104. Upon induction of bacterial cultures with arabinose,gelonin polypeptide could be detected in the culture supernatants withgelonin-specific antibodies. There were no differences detected in theexpression levels of gelonin from plasmids pING3734 and pING3825, or inthe levels from any of the gelonin variants. Each protein was producedin E. coli at levels approaching 1 g/l.

Reticulocyte Lysate Assay

The ability of gelonin and recombinant gelonin analogs to inhibitprotein synthesis in vitro was tested using a reticulocyte lysate assay(RLA) described in Press et al., Immunol. Letters, 14, 37-41 (1986). Theassay measures the inhibition of protein synthesis in a cell-free systemusing endogenous globin mRNA from a rabbit red blood cell lysate.Decreased incorporation of tritiated leucine (³ H-Leu) was measured as afunction of toxin concentration. Serial log dilutions of standard toxin(the 30 kD form of ricin A-chain, abbreviated as RTA 30), nativegelonin, recombinant gelonin (rGelonin) and gelonin analogs were testedover a range of 1 μg/ml to 1 pg/ml. Samples were tested in triplicate,prepared on ice, incubated for 30 minutes at 37° C., and then counted onan Inotec Trace 96 cascade ionization counter. By comparison with anuninhibited sample, the picomolar concentration of toxin (pM) whichcorresponds to 50% inhibition of protein synthesis (IC₅₀) wascalculated. As is shown in Table 2 below, recombinant gelonin and mostof its analogs exhibit activity in the RLA comparable to that of nativegelonin. For some of the analogs (such as Gels39), RLA activity wasdiminished.

                  TABLE 1                                                         ______________________________________                                               Toxin  IC.sub.50 (pM)                                                  ______________________________________                                               RTA 30 2.5                                                                    Gelonin                                                                              15                                                                     rGelonin                                                                             11                                                                     Gel.sub.C10                                                                          60                                                                     Gel.sub.C44                                                                          20                                                                     Gel.sub.C239                                                                         955                                                                    Gel.sub.C244                                                                         32                                                                     Gel.sub.C247                                                                         12                                                                     Gel.sub.C248                                                                         47                                                              ______________________________________                                    

Gel_(C60) and the gelonin analog with both native cysteines replacedwith alanines were both active in the RLA (data not shown).

Preparation of Gelonin Immunoconjugates

Gelonin analogs of the invention were variously conjugated to murine(A.T.C.C. HB9286) and chimeric H65 antibody and H65 antibody domains(including Fab, Fab' and F(ab')₂ fragments) specifically reactive withthe human T cell determinant CD5. H65 antibody was prepared and purifiedaccording to U.S. patent application Ser. No. 07/306,433, supra.Chimeric H65 antibody was prepared according to methods similar to thosedescribed in Robinson et al., Human Antibodies and Hybridomas, 2, 84-93(1991).

(1) Conjugation to H65 antibodies

To expose a reactive sulfhydryl, the unpaired cysteine residues of thegelonin analogs were first reduced by incubation with 0.1 to 2 mM DTT(30-60 minutes at room temperature), and then were desalted bysize-exclusion chromatography.

Specifically, the Gel_(C248) analog (3.8 mg/ml) was treated with 2 mMDTT for 60 minutes in 0.1 M NaPhosphate, 0.25 M NaCl, pH 7.5 buffer. TheGel_(C244) variant (7.6 mg/ml) was treated with 2 mM DTT for 30 minutesin 0.1 M NaPhosphate, 0.25 M NaCl, pH 7.5 buffer. The Gel_(C247) analog(4 mg/ml) was treated with 2 mM DTT for 30 minutes in 0.1 M NaPhosphate,0.5 M NaCl, pH 7.5 buffer with 0.5 mM EDTA. The Gel_(C239) variant (3.2mg/ml) was treated with 2 mM DTT for 30 minutes in 0.1 m NaPhosphate,0.5 M NaCl, pH 7.5 buffer with 0.5 mM EDTA. The Gel_(C44) analog (4.2mg/ml) was treated with 0.1 mM DTT for 30 minutes in 0.1 M NaPhosphate,0.1 M NaCl, pH 7.5 buffer with 0.5 mM EDTA. Lastly, the Gel_(C10)variant (3.1 mg/ml) was treated with 1 mM DTT for 20 minutes in 0.1 MNaPhosphate, 0.1 M NaCl, pH 7.5 buffer with 1 mM EDTA.

The presence of a free sulfhydryl was verified by reaction with DTNB andthe average value obtained was 1.4±0.65 SH/molecule. No free thiols weredetected in the absence of reduction.

H65 antibody and chimeric H65 antibody was chemically modified with thehindered linker 5-methyl-2-iminothiolane (M2IT) at lysine residues tointroduce a reactive sulfhydryl group as described in Goff et al.,Bioconjugate Chem., 1, 381-386 (1990).

Specifically, for conjugation with Gel_(C248) and Gel_(C244), murine H65antibody at 4 mg/mL was derivitized with 18x M2IT and 2.5 mM DTNB in 25mM TEOA, 150 mM NaCl, pH 8 buffer for 1 hour at 23° C. The reaction gave1.9 linkers per antibody as determined by DTNB assay.

For conjugation with Gel_(C247) and Gel_(C239), H65 antibody at 4.7mg/mL was derivitized with 20x M2IT and 2.5 mM DTNB in 25 mM TEOA 150 mMNaCl, pH 8 buffer for 50 minutes at 23° C. The reaction gave 1.6 linkersper antibody as determined by DTNB assay.

Before reaction with Gel_(C44), H65 antibody at 5.8 mg/mL wasderivitized with 20x m2IT and 2.5 mM DTNB in 25 mM TEOA, 150 mM NaCl, pH8 buffer for 30 minutes at 23° C. The reaction gave 1.5 linkers perantibody as determined by DTNB assay.

For conjugation with Gel_(C10), H65 antibody at 2.2 mg/mL wasderivitized with 10x m2IT and 2.5 mM DTNB in 25 mM TEOA, 150 mM NaCl, pH8 buffer for 1 hour at 23° C. The reaction gave 1.4 linkers per antibodyas determined by DTNB assay.

Chimeric H65 antibody was prepared for conjugation in a similar mannerto murine H65 antibody.

Two methods were initially compared for their effectiveness in preparingimmunoconjugates with recombinant gelonin. First, the native disulfidebond in recombinant gelonin was reduced by the addition of 2 mM DTT atroom temperature for 30 minutes. The reduced gelonin was recovered bysize-exclusion chromatography on a column of Sephadex GF-05LS andassayed for the presence of free sulfhydryls by the DTNB assay. 1.4 freeSH groups were detected. This reduced gelonin was then reacted withH65-(M2IT)-S-S-TNB (1.8 TNB groups/H65). Under these experimentalconditions, little or no conjugate was prepared between reduced geloninand thiol-activated H65 antibody.

In contrast, when both the recombinant gelonin and the H65 antibody werefirst derivitized with the crosslinker M2IT (creating gelonin-(M2IT)-SHand H65-(M2IT)-S-S-TNB) and then mixed together,H65-(M2IT)-S-S-(M2IT)-gelonin conjugate was prepared in good yield(toxin/antibody ratio of 1.6). The starting materials for thisconjugation (gelonin-(M2IT)-SH and H65-(M2IT)-S-S-TNB) containedlinker/protein ratios of 1.2 and 1.4, respectively.

The reduced gelonin analogs were mixed with H65-(M2IT)-S-S-TNB to allowconjugation. The following conjugation reactions were set up for eachanalog: 23 mg (in 7.2 ml) of H65-M2IT-TNB were mixed with a 5-fold molarexcess of Gels4_(s) (23 mg in 6 ml) for 2 hours at room temperature,then for 18 hours overnight at 4° C.; 23 mg (in 7.3 ml) of H65-m2IT-TNBwere mixed with a 5fold molar excess of Gel_(C244) (23 mg in 3 ml) for 3hours at room temperature, then for 18 hours overnight at 4° C.; 9 mg(in 2.8 mL) of H65-m2IT-TNB were mixed with a 5-fold molar excess ofGel_(C247) (9 mg in 2.25 mL) for 2 hours at room temperature, then for 5nights at 4° C.; 9 mg (in 2.8 mL) of H65-m2IT-TNB were mixed with a5-fold molar excess of Gels39 (9 mg in 2.6 mL) for 2 hours at roomtemperature, then at 4° C. for 3 days; 12 mg (in 1.9 mL) of H65-m2IT-TNBwere mixed with a 5.6-fold molar excess of Gel_(C44) (13.44 mg in 3.2mL) for 4.5 hours at room temperature, then 4° C. overnight; and 11 mgof H65-m2IT-TNB were mixed with a 5-fold molar excess of Gel_(C10) (11mg in 3.5 mL) for 4 hours at room temperature, then at 4° C. overnight.

Following conjugation, unreacted M2IT linkers on the antibody werequenched with 1:1 mole cysteamine to linker for 15 minutes at roomtemperature. The quenched reaction solution was then loaded onto a gelfiltration column [Sephadex G-150 (Pharmacia) in the case of Gel_(C248),Gel_(C247), Gel_(C244) and Gel₂₃₉ and an AcA-44 column (IBF Biotecnics,France) in the case of Gel_(C44) and Gel_(C10) ]. The reactions were runover the gel filtration columns and eluted with 10 mM Tris, 0.15M NaClpH 7. The first peak off each column was loaded onto BlueToyopearl®resin (TosoHaas, Philadelphia, Pa.) in 10 mM Tris, 30 mM NaCl,pH 7 and the product was eluted with 10 mM Tris, 0.5 M NaCl, pH 7.5.

Samples of the final conjugation products were run on 5% non-reduced SDSPAGE, Coomassie stained and scanned with a Shimadzu laser densitometerto quantitate the number of toxins per antibody (T/A ratio). The yieldof final product for each analog conjugate was as follows: Gel_(C248),17 mg with a T/A ration of 1.6; Gel_(C247), 1.1 mg with a T/A ratio of1; Gel_(C244) , 4.5 mgs with a T/A ratio of 1.46; Gel_(C239), 2.9 mgwith a T/A ratio of 2.4; Gel_(C44), 7.3 mg with a T/A ratio of 1.22; andGel_(C10), 6.2 mg with a T/A ratio of 1.37. Conjugation efficiency(i.e., conversion of free antibody to immunoconjugate) was significantlygreater (˜80%) for some analogs (Gel_(C10), Gel_(C44), Gel_(C239),Gel_(C247), and Gel_(C248)) than for others (˜10%, Gel_(C244)).

Control immunoconjugates of H65 antibody and chimeric H65 antibody withnative and recombinant gelonin were prepared by similar procedures.

(2) Conjugation with antibody fragments

Analog Gel_(C247) was conjugated to various chimeric [cFab, cFab' andcF(ab')₂ ] and "human engineered" [he1 Fab, he1 Fab' and he1 F(ab')₂ ]antibody fragments. Chimeric H65 antibody fragments may be preparedaccording to the methods described in U.S. patent application Ser. No.07/714,175, supra. H65 variable regions used to encode the variableregions of H65 antibody fragments were human engineered (referring tothe replacement of selected murine-encoded amino acids to make the H65antibody sequences less immunogenic to humans) according to the methodsdescribed in co-pending, co-owned U.S. patent application Ser. No.07/808,454, now abandoned filed Dec. 13, 1991, which is incorporated byreference herein.

The H65 antibody fragments were conjugated to Gel_(C247) analogbasically as described below for conjugation of human engineered Fab andFab' fragments to Gel_(C247).

The he1 Fab was dialyzed into 25 mM TEOA buffer, 250 mM NaCl, pH 8 andthen concentrated to 6.8 mg/mL prior to derivitization with the M2ITcrosslinker. For the linker reaction, M2IT was used at 20-fold molarexcess, in the presence of 2.5 mM DTNB. The reaction was allowed toproceed for 30 minutes at room temperature, then desalted on GF05 (gelfiltration resin) and equilibrated in 0.1 M Na Phosphate, 0.2M NaCl, pH7.5. A linker number of 1.8 linkers per Fab was calculated based on theDTNB assay. The hel Fab-M2IT-TNB was concentrated to 3.7 mg/mL prior toconjugation with Gel_(C247).

Gel_(C247) at 12.8 mg/mL in 10 mM Na Phosphate, 0.3M NaCl, was treatedwith 1 mM DTT, 0.5 mM EDTA for 20 minutes at room temperature to exposea reactive sulfhydryl for conjugation and then was desalted on GF05 andequilibrated in 0.1 M Na Phosphate, 0.2 M NaCl, pH 7.5. Free thiolcontent was determined to be 0.74 moles of free SH per mole ofGel_(C247) using the DTNB assay. The gelonin was concentrated to 8.3mg/mL prior to conjugation with activated antibody.

The conjugation reaction between the free thiol on Gels47 and thederivitized hel Fab-M2IT-TNB, conditions were as follows. A 5-foldexcess of the gelonin analog was added to activated hel Fab-M2IT-TNB(both proteins were in 0.1M Na Phosphate, 0.2M NaCl, pH 7.5) and thereaction mixture was incubated for 3.5 hours at room temperature andthen overnight at 4° C. Following conjugation, untreated M2IT linkerswere quenched with 1:1 mole cysteamine to linker for 15 minutes at roomtemperature. The quenched reaction solution was loaded onto a gelfiltration column (G-75) equilibrated with 10 mM Tris, 150 mM NaCl, pH7. The first peak off this column was diluted to 30 mM NaCl with 10 mMTris, pH7 and loaded on Blue Toyopearl®. The product was eluted with 10mM Tris, 0.5 M NaCl, pH 7.5.

Similarly, the H65 hel Fab' fragment was dialyzed into 25 mM TEOAbuffer, 400 mM NaCl, pH 8 at 2.9 mg/mL prior to derivitization with theM2IT crosslinker. For the linker reaction, M2IT was used at 20-foldmolar excess, in the presence of 2.5 mM DTNB. The reaction was allowedto proceed for 1 hour at room temperature then it was desalted on GF05(gel filtration resin) and equilibrated in 0.1 M Na Phosphate, 0.2 MNaCl, pH 7.5. A linker number of 1.6 linkers per Fab' was calculatedbased on the DTNB assay. The hel Fab'-M2IT-TNB was concentrated to 3.7mg/mL prior to conjugation with Gel_(C247).

The Gel_(C247) at 77 mg/mL was diluted with in 10 mM Na Phosphate, 0.1 MNaCl to a concentration of 5 mg/mL, treated with 1 mM DTT, 0.5 mM EDTAfor 30 minutes at room temperature to expose a free thiol forconjugation and then was desalted on GF05 and equilibrated in 0.1 M NaPhosphate, 0.2 M NaCl, pH 7.5. Free thiol content was determined to be1.48 moles of free SH per mole of Gel_(C247) using the DTNB assay. TheGel_(C247) was concentrated to 10 mg/mL prior to conjugation withactivated hel Fab'-M2IT-TNB.

For the reaction between the free thiol on Gel_(C247) and thederivitized hel Fab'-M2IT-TNB, conditions were as follows. A 5.7-foldmolar excess of gelonin was added to activated hel Fab'-M2IT-TNB and thefinal salt concentration was adjusted to 0.25 M. The reaction mix wasincubated for 1.5 hours at room temperature and then over the weekend at4° C. Following conjugation, unreacted M2IT linkers were quenched with1:1 mole cysteamine to linker for 15 minutes at room temperature. Thequenched reaction solution was loaded onto a gel filtration column(AcA54) equilibrated with 10 mM Tris, 250 mM NaCl, pH 7.5. The firstpeak off this column was diluted to 20 mM NaCl with 10 mM Tris, pH 7 andloaded on Blue Toyopearl® which was equilibrated in 10 mM Tris, 20 mMNaCl, pH 7. The column was then washed with 10 mM Tris, 30 mM Nacl, pH7.5. The product was eluted with 10 mM Tris, 1 M NaCl, pH 7.5.

Whole Cell Kill Assays

Immunoconjugates prepared with gelonin and gelonin analogs were testedfor cytotoxicity against an acute lymphoblastoid leukemia T cell line(HSB2 cells) and against human peripheral blood mononuclear cells(PBMCs). Immunoconjugates of ricin A-chain with H65 antibody (H65-RTA)and antibody fragments were also tested. The ricin A-chain (RTA) as wellas the H65-RTA immunoconjugates were prepared and purified according toU.S. patent application Ser. No. 07/306,433, supra.

Briefly, HSB2 cells were incubated with immunotoxin and the inhibitionof protein synthesis in the presence of immunotoxin was measuredrelative to untreated control cells. The standard immunoconjugatesH65-RTA (H65 derivatized with SPDP linked to RTA), H65-Gelonin andH65-rGelonin, H65 fragment immunoconjugate, and gelonin immunoconjugatesamples were diluted with RPMI without leucine at half-logconcentrations ranging from 2000 to 0.632 ng/ml. All dilutions wereadded in triplicate to microtiter plates containing 1×10⁵ HSB2 cells.HSB2 plates were incubated for 20 hours at 37° C. and then pulsed with ³H-Leu for 4 hours before harvesting. Samples were counted on the InotecTrace 96 cascade ionization counter. By comparison with an untreatedsample, the picomolar concentration (pM) of immunotoxin which resultedin a 50% inhibition of protein synthesis (IC₅₀) was calculated. In orderto normalize for conjugates containing differing amounts of toxin ortoxin analog, the cytotoxicity data were converted to picomolar toxin(pM T) by multiplying the conjugate IC₅₀ (in pM) by the toxin/antibodyratio which is unique to each conjugate preparation.

The PMBC assays were performed as described by Fishwild et al., Clin.and Exp. Immunol., 86, 506-513 (1991) and involved the incubation ofimmunoconjugates with PBMCs for a total of 90 hours. During the final 16hours of incubation, ³ H-thymidine was added; upon completion,immunoconjugate-induced inhibition of DNA synthesis was quantified. Theactivities of the H65 and chimeric H65 antibody conjugates against HSB2cells and PBMC cells are listed in Table 2 below.

                  TABLE 2                                                         ______________________________________                                                            IC.sub.50 (pM T)                                                                HSB2                                                    Conjugate             Cells        PBMCs                                      ______________________________________                                        H65-RTA               143          459                                        H65-(M2IT)-S-S-(M2IT)-Gelonin                                                                       1770         81                                         H65-(M2IT)-S-S-(M2IT)-rGelonin                                                                      276          75                                         H65-(M2IT)-S-S-Gel.sub.C10                                                                          140          28                                         H65-(M2IT)-S-S-Gel.sub.C44                                                                          99           51                                         H65-(M2IT)-S-S-Gel.sub.C239                                                                         2328         180                                        H65-(M2IT)-S-S-Gel.sub.C244                                                                         >5000        >2700                                      H65-(M2IT)-S-S-Gel.sub.C247                                                                         41           35                                         H65-(M2IT)-S-S-Gel.sub.C248                                                                         440          203                                        cH65-RTA.sub.30       60           400                                        cH65-(M2IT)-S-S-(M2IT)-Gelonin                                                                      1770         140                                        cH65-(M2IT)-S-S-(M2IT)-rGelonin                                                                     153          120                                        cH65-(M2IT)-S-S-Gel.sub.C239                                                                        >7000        290                                        cH65-(M2IT)-S-S-Gel.sub.C247                                                                        34           60                                         cH65-(M2IT)-S-S-Gel.sub.C248                                                                        238          860                                        ______________________________________                                    

Against HSB2 cells, many of the gelonin analog immunoconjugates weresignificantly more potent than conjugates prepared with native geloninor recombinant, unmodified gelonin, both in terms of a low IC₅₀ value,but also in terms of a greater extent of cell kill. Against human PBMCs,the gelonin analog conjugates were at least as active as native andrecombinant gelonin. Importantly, however, some of the conjugates (suchas Gel_(C10) Gel_(C10), and Gel_(C247)) exhibited an enhanced potencyagainst PBMCs, and also exhibited an enhanced level of cell kill (datanot shown).

The activities of the H65 antibody fragment conjugates against HSB2cells and PBMC cells are listed in Tables 3 and 4 below, wherein extentof kill in Table 4 refers to the percentage of protein synthesisinhibited in HSB2 cells at the highest immunotoxin concentration tested(1 μg/ml).

                  TABLE 3                                                         ______________________________________                                                       IC.sub.50 (pM T)                                               Conjugate        HSB2 Cells                                                                              PBMCs                                              ______________________________________                                        cFab'-RTA 30     530       1800                                               cFab'-rGelonin   135       160                                                cFab'-Gel.sub.C247                                                                             48        64                                                 cF(ab').sub.2 -RTA 30                                                                          33        57                                                 cF(ab').sub.2 -rGelonin                                                                        55        34                                                 cF(ab').sub.2 -Gel.sub.C247                                                                    23        20                                                 cF(ab').sub.2 -Gel.sub.C248                                                                    181       95                                                 ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                                      IC.sub.50 (pM T)                                                Conjugate       HSB2 Cells                                                                              Extent of Kill                                      ______________________________________                                        he1 Fab'-Gel.sub.C247                                                                         57.7      93%                                                 he1 Fab-Gel.sub.247                                                                           180       94%                                                 cFab'-Gel.sub.C247                                                                            47.5      93%                                                 cF(ab').sub.2 -rGelonin                                                                       45.4      85%                                                 mF(ab').sub.2 -Gel.sub.C247                                                                   77.5      83%                                                 cF(ab').sub.2 -Gel.sub.C247                                                                   23.2      85%                                                 ______________________________________                                    

The cFab'-Gel₂₄₇ immunoconjugate is clearly more cytotoxic than cFab'conjugates with recombinant gelonin or RTA 30.

Solubility

Gelonin analogs and gelonin immunoconjugates exhibited enhancedsolubility in comparison to recombinant gelonin, native RTA 30 andimmunoconjugates of recombinant gelonin and RTA 30.

Disulfide Bond Stability Assay

The stability of the disulfide bond linking a RIP to a targetingmolecule (such as an antibody) is known to influence the lifespan ofimmunoconjugates in vivo [see Thorpe et al., Cancer Res., 47, 5924-5931(1987)]. For example, conjugates in which the disulfide bond is easilybroken by reduction are less stable and less efficacous in animal models[see Thorpe et al., Cancer Res., 48, 6396-6403 (1988)].

Immunoconjugates prepared with native gelonin, recombinant gelonin andgelonin analogs were therefore examined in an in vitro disulfide bondstability assay similar to that described in Wawrzynczak et al., CancerRes., 50, 7519-7526 (1990). Conjugates were incubated with increasingconcentrations of glutathione for 1 hour at 37° C. and, afterterminating the reaction with iodoacetamide, the amount of RIP releasedwas quantitated by size-exclusion HPLC on a TosoHaas TSK-G2000SW column.

By comparison with the amount of RIP released by high concentrations of2-mercaptoethanol (to determine 100% release), the concentration ofglutathione required to release 50% of the RIP (the RC₅₀) wascalculated. The results of assays for H65 antibody conjugates are setout in Table 5 below, wherein multiple RC₅₀ results or ranges given fora

                  TABLE 5                                                         ______________________________________                                        Conjugate             RC.sub.50 (mM)                                          ______________________________________                                        H65-RTA 30            3.2                                                     H65-(M2IT)-S-S-(M2IT)-gelonin                                                                       11.1                                                    H65-(M2IT)-S-S-(M2IT)-rGelonin                                                                      3.0                                                     H65-(M2IT)-S-S-Gel.sub.C.sub.10                                                                     2.5                                                     H65-(M2IT)-S-S-Gel.sub.C44                                                                          0.6                                                     H65-(M2IT)-S-S-Gel.sub.C239                                                                         774.0                                                   H65-(M2IT)-S-S-Gel.sub.C244                                                                         1.2                                                     H65-(M2IT)-S-S-Gel.sub.C247                                                                         0.1                                                     H65-(M2IT)-S-S-Gel.sub.C248                                                                         0.4                                                     cH65-RTA 30           2.50                                                    cH65-(M2IT)-S-S-(M2IT)-rGelonin                                                                     2.39                                                    cH65-(M2IT)-S-S-Gel.sub.C247                                                                        0.11                                                    cH65-(M2IT)-S-S-Gel.sub.C248                                                                        0.32                                                    ______________________________________                                    

The foregoing results indicate that the stability of the bonds betweenthe different gelonin proteins and H65 antibody varied greatly. With theexception of Gel_(C10) and Gel_(C239), most of the gelonin analogsresulted in conjugates with linkages that were somewhat less stable inthis in vitro assay than the dual-linker chemical conjugate. Thestability of the Gel_(C239) analog, however, was particularly enhanced.

The results of the assay for H65 antibody fragment conjugates are setout in Table 6 below.

                  TABLE 6                                                         ______________________________________                                        Conjugate       RC.sub.50 (mM)                                                ______________________________________                                        he1 Fab'-Gel.sub.C247                                                                         0.07                                                          cFab'-Gelonin   1.27                                                          cFab'-Gel.sub.C247                                                                            0.08                                                          cF(ab').sub.2 -RTA 30                                                                         3.69                                                          cF(ab').sub.2 -rGelonin                                                                       2.30                                                          cF(ab').sub.2 -Gel.sub.C247                                                                   0.09                                                          cF(ab').sub.2 -Gel.sub.C248                                                                   0.32                                                          ______________________________________                                    

From the RC₅₀ results presented in Tables 5 and 6, it appears that theparticular RIP analog component of each immunotoxin dictates thestability of the immunotoxin disulfide bond in vitro.

Example 2

BRIP possesses characteristics which make it an attractive candidate fora component of immunotoxins. BRIP is a naturally unglycosylated proteinthat may have reduced uptake in the liver and enhanced circulatoryresidence time in vivo. Additionally, BRIP is less toxic and lessimmunogenic in animals than the A-chain of ricin. Cloning of the BRIPgene and expression of recombinant BRIP in an E. coli expression systemobviates the need to purify native BRIP directly from barley, andenables the development of analogs of BRIP which may be conjugated withan available cysteine residue for conjugation to antibodies.

Purification of BRIP and Generation of Polyclonal Antibodies to BRIP

Native BRIP was purified from pearled barley flour. Four kilograms offlour was extracted with 16 liters of extraction buffer (10 mM NAPO4, 25mM NaCl, pH 7.2) for 20 hours at 4° C. The sediment was removed bycentrifugation, and 200 ml of packed S-Sepharose (Pharmacia, Piscataway,N.J.) was added to absorb BRIP. After mixing for 20 hours at 4° C., theresin was allowed to settle out, rinsed several times with extractionbuffer and then packed into a 2.6×40 cm column. Once packed, the columnwas washed with extraction buffer (150 ml/h) until the absorbance of theeffluent approached zero. BRIP was then eluted with a linear gradient of0.025 to 0.3 M NaCl in extraction buffer and 5 ml fractions werecollected. BRIP-containing peaks (identified by Western analysis ofcolumn fractions) were pooled, concentrated to about 20 ml, and thenchromatographed on a 2.6×100 cm Sephacryl S-200HR (Pharmacia) columnequilibrated in 10 mM NaPO₄, 125 mM NaCl, pH 7.4 (10 ml/hr).BRIP-containing peaks were pooled again, concentrated, and stored at-70° C.

The resulting purified BRIP protein had a molecular weight of about30,000 Daltons, based upon the mobility of Coomassie-stained proteinbands following SDS-PAGE. The amino acid composition was consistent withthat published by Asano et al., Carlsberg Res. Comm., 49, 619-626(1984).

Rabbits were immunized with purified BRIP to generate polyclonalantisera.

Cloning of the BRIP Gene

A cDNA expression library prepared from germinating barley seeds in thephage λ expression vector λZAPII was purchased from Stratagene, LaJolla, Calif. Approximately 700,000 phage plaques were screened withanti-BRIP polyclonal antisera and 6 immunoreactive plaques wereidentified. One plaque was chosen, and the cDNA contained therein wasexcised from λZAPII with EcoRI and subcloned into pUC18 generating thevector pBS1. The cDNA insert was sequenced with Sequenase (United StatesBiochemical, Cleveland, Ohio). The DNA sequence of the native BRIP geneis set out in SEQ ID NO: 12 and in FIG. 11. To confirm that cDNA encodedthe native BRIP gene, the cDNA was expressed in the E. coli plasmidpKK233-2 (Pharmacia). BRIP protein was detected in IPTG-induced cellstransformed with the plasmid by Western analysis with above-describedrabbit anti-BRIP antisera.

Construction of an E. coli Expression Vector Containing the BRIP Gene

Barley cDNA containing the BRIP gene was linked to a pelB leadersequence and placed under control of an araB promoter in a bacterialsecretion vector.

An intermediate vector containing the BRIP gene linked to the pelBleader sequence was generated. Plasmid pBS1 was cut with NcoI, treatedwith Mung Bean Nuclease, cut with BamHI and the 760 bp fragmentcorresponding to amino acids 1-256 of BRIP was purified from an agarosegel. Concurrently, a unique XhoI site was introduced downstream of the3'-end of the BRIP gene in pBS1 by PCR amplification with a pUC18 vectorprimer (identical to the Reverse® primer sold by NEB or BRL butsynthesized on a Cyclone Model 8400 DNA synthesizer) and the specificprimer BRIP 3'Xho. The sequence of each of the primers is set out below.##STR12## Primer BRIP 3'Xho includes a portion corresponding to the last8 bp of the BRIP gene, the termination codon and several base pairsdownstream of the BRIP gene, and an additional portion that introduces aXhoI site in the resulting PCR fragment. The PCR reaction product wasdigested with BamHI and XhoI, and an 87 bp fragment containing the3'-end of the BRIP gene was purified on a 5% acrylamide gel. The 760 and87 bp purified BRIP fragments were ligated in the vector pING1500adjacent to the PelB leader sequence. pING1500 had previously been cutwith SstI, treated with T4 polymerase, cut with XhoI, and purified. TheDNA sequence at the junction of the pelB leader and the 5'-end of theBRIP gene was verified by DNA sequence analysis. This vector was denotedpING3321-1.

The final expression vector was assembled by placing the BRIP gene underthe control of the inducible araB promoter. Plasmid pING3321-1 was cutwith PstI and XhoI, and the BRIP gene linked to the pelB leader waspurified from an agarose gel. The expression vector pING3217, containingthe araB promoter, was cut with PstI and XhoI and ligated to the BRIPgene. The expression vector was denoted pING3322.

Arabinose induction of E. coli cells containing the plasmid pING3322 ina fermenter resulted in the production of about 100 mg per liter ofrecombinant BRIP. E. coli-produced BRIP displays properties identical toBRIP purified directly from barley seeds.

Construction of BRIP Analogs With a Free Cysteine Residue

The BRIP protein contains no cysteine residues, and therefore containsno residues directly available which may form a disulfide linkage toantibodies or other proteins. Analogs of recombinant BRIP were generatedwhich contain a free cysteine residue near the C-terminus of theprotein. Three residues of the BRIP protein were targets for amino acidsubstitutions. Comparison of the amino acid sequence of BRIP to theknown tertiary structure of the ricin A-chain (see FIG. 2) suggestedthat the three positions would be available near the surface of themolecule. The three BRIP analogs include cysteines substituted in placeof serine₂₇₇, alanine₂₇₀, and leucine₂₅₆ of the native protein, and weredesignated BRIP_(C277), BRIP_(C270) and BRIP_(C256), respectively.

(1) A plasmid vector capable of expressing the BRIP_(C277) analog wasconstructed by replacing the 3'-end of the BRIP gene with a DNA segmentconferring the amino acid change. The EcoRI fragment containing the BRIPgene from pBS1 was subcloned into M13mp18, and single-stranded DNA(anti-sense strand) was amplified by PCR with primers OBM2(corresponding nucleotides -11 to +8 of the BRIP gene) and OMB4(corresponding to amino acids 264-280 of BRIP and the termination codonof BRIP, and incorporating the substitution of a cysteine codon for thenative codon for serine₂₇₇ of native BRIP). The sequences of primersOBM2 and OMB4, wherein the underlined nucleotides encode the substitutedcysteine, are set out below. ##STR13## A fragment containing a BRIP genein which the codon for the amino acid at position 277 was changed to acysteine codon was amplified. The fragment was cloned into the SmaI siteof pUC19 (BRL) and the plasmid generated was denoted pMB22. pMB22 wasdigested with EcoRI and an EcoRI-XhoI linker (Clonetech, Palo Alto,Calif.) was ligated into the vector. Subsequent digestion with XhoI andreligation generated vector pINGMB2X. A BamHI to XhoI fragment encodingthe 3'-end of BRIP with the altered amino acid was excised from pMB2Xand the fragment was purified on a 5% acrylamide gel. This fragmentalong with an EcoRI to BamHI fragment containing the pelB leadersequence and sequences encoding the first 256 amino acids of BRIP weresubstituted in a three piece ligation into pING3322 cut with EcoRI andXhoI. The resulting vector containing the BRIP_(C277) analog wasdesignated. pING3803 (A.T.C.C. Accession No. 68722).

(2) A BRIP analog with a free cysteine at position 256 was constructedusing PCR to introduce the amino acid substitution. A portion of theexpression plasmid pING3322 was amplified with primers BRIP-256 andHindIII-2. The sequence of each primer is set out below. ##STR14##Nucleotides 4-21 of primer BRIP-256 encode amino acids 256-262 of BRIPwhile the underlined nucleotides specify the cysteine to be substitutedfor the leucine at the corresponding position of the native BRIPprotein. Primer HindIII-2 corresponds to a portion of the plasmid. ThePCR product, which encodes the carboxyl terminal portion of the BRIPanalog, was treated with T4 polymerase, cut with XhoI, and the resultingfragment was purified on a 5% acrylamide gel. Concurrently, plasmidpING3322 was cut with BamHI, treated with T4 polymerase, cut with EcoRI,and the fragment containing the pelB leader sequence and sequencesencoding the first 256 amino acids of BRIP was purified. The twofragments were then assembled back into pING3322 to generate the geneencoding the analog BRIP_(C256). This plasmid is denoted pING3801.

(3) A BRIP analog with a cysteine at position 270 was also generatedusing PCR. A portion of the expression plasmid pING3322 was amplifiedwith primers BRIP-270 and the HindIII-2 primer (SEQ ID NO: 50). Thesequence of primer BRIP-270 is set out below. ##STR15## Primer BRIP-270corresponds to amino acids 268-276 of BRIP with the exception of residue270. The codon of the primer corresponding to position 270 specifies acysteine instead of the alanine present in the corresponding position innative BRIP. The PCR product was treated with T4 polymerase, cut withXhoI, and the 51 bp fragment, which encodes the carboxyl terminalportion of the analog, was purified on a 5% acrylamide gel. The fragment(corresponding to amino acids 268-276 of BRIP_(C270)) was cloned in athree piece ligation along with the internal 151 bp BRIP restrictionfragment from SstII to MscI (corresponding to BRIP amino acids 217-267)from plasmid pING3322, and restriction fragment from SstII to XhoI frompING3322 containing the remainder of the BRIP gene. The plasmidgenerated contains the gene encoding the BRIP_(C270) analog and isdesignated pING3802.

Purification of Recombinant BRIP and the BRIP Analogs

Recombinant BRIP (rBRIP) and the BRIP analogs with free cysteineresidues were purified essentially as described for native BRIP exceptthey were prepared from concentrated fermentation broths. For rBRIP,concentrated broth from a 10 liter fermentation batch was exchanged into10 mM Tris, 20 mM NaCl pH 7.5, loaded onto a Sephacryl S-200 column, andeluted with a 20 to 500 mM NaCl linear gradient. Pooled rBRIP wasfurther purified on a Blue Toyopearl® column (TosoHaas) loaded in 20 mMNaCl and eluted in a 20 to 500 mM NaCl gradient in 10mM Tris, pH 7.5.For BRIP analogs, concentrated fermentation broths were loaded onto aCM52 column (Whatman) in 10 mM phosphate buffer, pH 7.5, and eluted witha 0 to 0.3M NaCl linear gradient. Further purification was bychromatography on a Blue Toyopearl® column.

Reticulocyte Lysate Assay

The ability of the rBRIP and the BRIP analogs to inhibit proteinsynthesis in vitro was tested by reticulocyte lysate assay as describedin Example 1. Serial log dilutions of standard toxin (RTA 30), nativeBRIP, rBRIP and BRIP analogs were tested over a range of 1 μg/ml to 1pg/ml. By comparison with an uninhibited sample, the picomolarconcentration of toxin (pM) which corresponds to 50% inhibition ofprotein synthesis (IC₅₀) was calculated. The results of the assays arepresented below in Table 7.

                  TABLE 7                                                         ______________________________________                                        Toxin          IC.sub.50 (pM)                                                 ______________________________________                                        RTA 30         3.1                                                            Native BRIP    15                                                             rBRIP          18                                                             BRIP.sub.C256  23                                                             BRIP.sub.C270  20                                                             BRIP.sub.C277  24                                                             ______________________________________                                    

The RLA results indicate that the BRIP analogs exhibitribosome-inactivating activity comparable to that of the recombinant andnative BRIP toxin. All the analogs retained the natural ability ofnative BRIP to inhibit protein synthesis, suggesting that amino acidsubstitution at these positions does not affect protein folding andactivity.

Construction of BRIP Immunoconjugates

Immunoconjugates of native BRIP with 4A2 (described in Morishima et al.,J. Immunol., 129, 1091 (1982) and H65 antibody (obtained from hybridomaA.T.C.C. HB9286) which recognize the T-cell determinants CD7 and CD5,respectively, were constructed. Immunoconjugates of ricin A-chains(RTAs) with 4A2 and H65 antibody were constructed as controls. The H65antibody and ricin A-chains as well as the RTA immunoconjugates wereprepared and purified according to U.S. patent application Ser. No.08/130,406 supra.

To prepare immunoconjugates of native BRIP, both the antibody (4A2 orH65) and native BRIP were chemically modified with the hindered linker5-methyl-2-iminothiolane (M2IT) at lysine residues to introduce areactive sulfhydryl group as described in Goff et al., BioconjugateChem., 1, 381-386 (1990). BRIP (3 mg/ml) was first incubated with 0.5 mMM2IT and 1 mM DTNB in 25 mM triethanolamine, 150 mM NaCl, pH 8.0, for 3hours at 25° C. The derivatized BRIP-(M2IT)-S-S-TNB was then desalted ona column of Sephadex GF-05LS and the number of thiol groups introducedwas quantitated by the addition of 0.1 mM DTT. On average, each BRIPmolecule contained 0.7 SH/mol.

4A2 or H65 antibody (4 mg/ml) in triethanolamine buffer was similarlyincubated with M2IT (0.3 mM) and DTNB (1 mM) for 3 hours at 25° C.Antibody-(M2IT)-S-S-TNB was then desalted and the TNB:antibody ratio wasdetermined. To prepare the conjugate, the BRIP-(M2IT)-S-S-TNB was firstreduced to BRIP-(M2IT)-SH by treatment with 0.5 mM DTT for 1 hour at 25°C., desalted by gel filtration of Sephadex® GF-05LS to remove thereducing agent, and then mixed with antibody-(M2IT)-S-S-TNB.

Following a 3 hour incubation at 25° C., and an additional 18 hours at4° C., the conjugate was purified by sequential chromatography on AcA44(IBF) and Blue Toyopearl®. Samples of the final product were run on 5%non-reducing SDS PAGE, Coomassie stained, and scanned with a Shimadzulaser densitometer to quantitate the number of toxins per antibody.

The BRIP analogs containing a free cysteine were also conjugated to 4A2and H65 antibodies. The analogs were treated with 50 mM DTT either for 2hours at 25° C. or for 18 hours at 4° C. to expose the reactivesulfhydryl group of the cysteine and desalted. The presence of a freesulfhydryl was verified by reaction with DTNB [Ellman et al., Arch.Biochem. Biophys, 82, 70-77 (1959)]. 4A2 or H65 antibody derivatized asdescribed above with M2IT was incubated with the reduced BRIP analogs ata ratio of 1:5 at room temperature for 3 hours and then overnight at 4°C. Immunoconjugates H65-BRIP_(C256), 4A2-BRIP_(C256), H65-BRIP_(C277)were prepared in 25 mM triethanolamine, 150 mM NaCl pH 8, whileimmunoconjugates H65-BRIP_(C270), 4A2-BRIP_(C270) and 4A2-BRIP_(C277)were prepared in 0.1 M sodium phosphate, 150 mM NaCl pH 7.5. Followingconjugation, 10 μM mercaptoethylamine was added for 15 minutes at 25° C.to quenched any unreacted m2IT linkers on the antibody. The quenchedreaction solution was promptly loaded onto a gel filtration column(AcA44) to remove unconjugated ribosome-inactivating protein.Purification was completed using soft gel affinity chromatography onBlue Toyopearl® resin using a method similar to Knowles et al., Analyt.Biochem., 160, 440 (1987). Samples of the final product were run on 5%non-reduced SDS PAGE, Coomassie stained, and scanned with a Shimadzulaser densitometer to quantitate the number of toxins per antibody. Theconjugation efficiency was substantially greater for BRIP_(C277) (78%)than for either of the other two analogs, BRIP_(C270) and BRIP_(C256)(each of these was about 10%). Additionally, the BRIP_(C277) product wasa polyconjugate, i.e., several BRIP molecules conjugated to a singleantibody, in contrast to the BRIP_(C270) and BRIP_(C256) products whichwere monoconjugates.

Whole Cell Kill Assay

Immunoconjugates of native BRIP and of the BRIP analogs were tested forthe ability to inhibit protein synthesis in HSB2 cells by the whole cellkill assay described in Example 1. Standard immunoconjugates H65-RTA(H65 derivatized with SPDP linked to RTA) and 4MRTA (4A2 antibodyderivatized with M2IT linked to RTA) and BRIP immunoconjugate sampleswere diluted with RPMI without leucine at half-log concentrationsranging from 2000 to 0.632 ng/ml. All dilutions were added in triplicateto microtiter plates containing 1×10⁵ HSB2 cells. HSB2 plates wereincubated for 20 hours at 37° C. and then pulsed with ³ H-Leu for 4hours before harvesting. Samples were counted on the Inotec Trace 96cascade ionization counter. By comparison with an untreated sample, thepicomolar toxin concentration (pM T) of immunoconjugate which resultedin a 50% inhibition of protein synthesis (IC₅₀) was calculated. Theassay results are presented below in Table 8.

                  TABLE 8                                                         ______________________________________                                        Conjugate      IC.sub.50 (pM T)                                               ______________________________________                                        4A2-BRIP       122.45                                                         4A2-BRIP.sub.C270                                                                            46.3                                                           4A2-BRIP.sub.C277                                                                            57.5                                                           4A2-BRIP.sub.C256                                                                            1116                                                           H65-BRIP       >5000                                                          H65-BRIP.sub.C277                                                                            1176                                                           ______________________________________                                    

The BRIP analog conjugates were less potent than the ricin conjugatecontrol (data not shown). The immunotoxins containing antibody 4A2 andeither the BRIP_(C270) or the BRIP_(C277) analog exhibited comparable toincreased specific cytotoxicity toward target cells as compared toimmunotoxin containing native BRIP. While 4A2-BRIP_(C256) is less activethan 4A2-BRIP, 4A2-BRIP_(C270) and 4A2-BRIP_(C277) are between 3 and 4times more active. Similarly, the immunoconjugate of H65 to BRIP_(C277)shows greater toxicity toward target cells than the immunoconjugate ofH65 to native BRIP. Thus, linkage of antibody to BRIP derivatives whichhave an available cysteine residue in an appropriate location results inimmunotoxins with enhanced specific toxicity toward target cellsrelative to conjugates with native BRIP.

Disulfide Bond Stability Assay

Immunoconjugates prepared with native BRIP and the BRIP analogs wereexamined by the disulfide bond stability assay described in Example 1.Briefly, conjugates were incubated with increasing concentrations ofglutathione for 1 hour at 37° C. and, after terminating the reactionwith iodoacetamide, the amount of RIP released was quantitated bysize-exclusion HPLC on a TosoHaas TSK-G2000SW column.

By comparisons with the amount of RIP released by high concentrations of2-mercaptoethanol (to determine 100% release), the concentration ofglutathione required to release 50% of the RIP (the RC₅₀) wascalculated. As shown below in Table 9, the conjugates prepared withBRIP_(C270) or BRIP_(C277) were significantly more stable than eitherthe RTA conjugates or those prepared with native BRIP.

                  TABLE 9                                                         ______________________________________                                        Conjugate       RC.sub.50 (mM)                                                ______________________________________                                        H65-RTA         7.0                                                           H65-BRIP        2.8                                                           H65-BRIP.sub.C277                                                                             196.0                                                         4A2-RTA         4.4                                                           4A2-BRIP        3.3                                                           4A2-BRIP.sub.C270                                                                             53.0                                                          4A2-BRIP.sub.C277                                                                             187.0                                                         ______________________________________                                    

These unexpected results suggest that conjugates prepared with Type IRIP analogs according to the present invention may have enhancedstability and efficacy in vivo.

Example 3

Plants of the genus Momordica produce a number of related proteins knownas momordins or momorcharins which are Type I RIPs. The gene encodingmomordin II was cloned from Momordica balsamina seeds.

Preparation of M. balsamina RNA

Total RNA was prepared from 4 g of M. balsamina seeds as described inAusubel et al., supra. PolyA containing RNA was prepared from 1 mg oftotal RNA by chromatography on oligo-(dT)-cellulose. 40 mg ofoligo-(dT)-cellulose Type 7 (Pharmacia) was added to 0.1 N NaOH andpoured into a disposable column (Biorad). The column was washed withwater until the eluate was pH 5.5, and then was washed with 1X loadingbuffer (50 mM NaCitrate, 0.5M NaCl, 1 mM EDTA, 0.1% SDS, pH 7.0) untilthe eluate was pH 7.0. 1 mg of total RNA was suspended in 300 μl ofwater, heated to 65° C. for 5 minutes, and 300 μl of 2X loading bufferwas added (100 mM Na Citrate, 1M NaCl, 2 mM EDTA, and 0.2% SDS). The RNAwas loaded onto the column, and the flow through was reheated to 65° C.,cooled to room temperature, and reloaded onto the column. Column-boundmRNA was washed 5 times with 0.5 ml of 1X loading buffer, and two timeswith 0.5 ml of 0.05M NaCitrate, 0.1 M NaCl, 1 mM EDTA, 0.1% SDS.PolyA-containing RNA was eluted two times from the column with 0.5 ml of25 mM NaCitrate, 1 mM EDTA, and 0.05% SDS.

Library Preparation

A cDNA library from the polyA-containing M. balsamina RNA was preparedin a bacterial expression plasmid with the SuperScript Plasmid System(BRL, Gaithersburg, Md.). The cDNA was synthesized from 2 μg of polyA-containing RNA, size fractionated, digested with NotI, and ligatedinto the expression vector pSPORT as recommended by the manufacturer ofthe vector, BRL.

Cloning of the Momordin II Gene

A DNA fragment encoding the first 27 amino acids of momordin II wasamplified from M. balsamina cDNA by PCR. First strand cDNA was preparedfrom 100 ng of polyA containing RNA with an RNA-PCR Kit (Perkin ElmerCetus). Two partially degenerate primers were synthesized based on theamino acid sequence of the first 27 amino acids of momordin II describedin Li et al., Experientia, 36, 524-527 (1980). Because the amino acidsequence of amino acids 1-27 of momordin II is 52% homologous to aminoacids 1-17 of momordin I [Ho et al., BBA, 1088, 311-314 (1991)], somecodon assignments in the degenerate primers were based on homology tothe corresponding amino acid as well as codon preference in the momordinI gene. The sequences of primers momo-3 and momo-4 are set out belowusing IUPAC nucleotide symbols. ##STR16## The resulting 81 bp PCRproduct was purified on a 5% acrylamide gel and cloned into the Smalsite of pUC18. Three candidate clones were sequenced, and one clone,pMO110, was identified which encoded the N-terminal 27 amino acids ofmomordin II.

A hybridization probe was designed for screening of the momordin II cDNAlibrary based on the sequence of the pMO110 momordinII DNA fragment. Thesequence of the primer momo-5 is shown below. ##STR17## Primer momo-5corresponds to amino acids 9-18 of mature momordin II. The underlinednucleotides of the primer were expected to match the DNA sequence of themomordin II gene exactly. Since this sequence is highly A/T-rich and mayhybridize to the momordin II gene weakly, the additional adjacentnucleotides were included in the primer. Bases 3 and 30 (overlined) werein the "wobble" position (i.e., the third nucleotide in a codon) ofamino acids 9 (alanine) and 18 (isoleucine), respectively, of momordinII and may not be identical to the nucleotide bases in the native gene.

A 90,000 member cDNA library in pSPORT was screened with ³² P-kinasedmomo-5, and eight potential candidate clones were identified. One clone,pING3619, was sequenced and contains an open reading frame correspondingin part to the expected N-terminal 27 residues of Momordin II. Thecomplete momordin gene contains 286 amino acids, the first 23 of whichare a presumed leader signal (mature momordin II is 263 residues). TheDNA sequence of the momordin II gene is set out in SEQ ID NO: 13 and inFIG. 12.

Construction of an Expression Vector Containing the Momordin II Gene

A bacterial expression vector for the momordin II gene was constructed.Two PCR primers were synthesized, one (momo-9) which primes from the +1residue of the mature momordin II amino acid sequence, and one at theC-terminus (momo-10) of momordin II which introduces an XhoI restrictionsite: ##STR18## pING3619 was amplified with momo-9 and momo-10, and theproduct was treated with T4 polymerase, cut with XhoI, and purified onan agarose gel. This gene fragment was ligated along with the 131 bppelB leader fragment from pIC100 which has been generated by SstIdigestion, T4-polymerase treatment, and EcoRI digestion, into the araBexpression vector cleaved with EcoRI and XhoI. The product of this threepiece ligation was sequenced to verify that the pelB junction andmomordin II coding sequence were correct. Arabinose induction of cellscontaining the momordin II expression plasmid pING3621 results inproduction of momordin II in E. coli.

Analogs of Mormordin II

Mormordin II has no natural cysteines available for conjugation toantibody. Analogs of momordin which have a free cysteine for conjugationto an antibody may be constructed. Positions likely to be appropriatefor substitution of a cysteine residue may be identified from FIG. 3 aspositions near the ricin A-chain cysteine₂₅₉ and as positions includingthe last 26 amino acids of momordin II that are accessible to solvent.For example, the arginine at position 242 of momordin II aligns with thericin A-chain cysteine at position 259 and is a preferred target forsubstitution. Additional preferred substitution positions for momordinII include the serine at position 241 and the alanine at position 243.

While the present invention has been described in terms of preferredembodiments, it is understood that variations and improvements willoccur to those skilled in the art. Therefore, it is intended that theappended claims cover all such equivalent variations which come withinthe scope of the invention as claimed.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 57                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 267 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       IlePheProLysGln TyrProIleIleAsnPheThrThrAlaGlyAla                             151015                                                                        ThrValGlnSerTyrThrAsnPheIleArgAlaValArgGlyArgLeu                              20 2530                                                                       ThrThrGlyAlaAspValArgHisGluIleProValLeuProAsnArg                              354045                                                                        ValGlyLeuProIleAsn GlnArgPheIleLeuValGluLeuSerAsn                             505560                                                                        HisAlaGluLeuSerValThrLeuAlaLeuAspValThrAsnAlaTyr                              6570 7580                                                                     ValValGlyTyrArgAlaGlyAsnSerAlaTyrPhePheHisProAsp                              859095                                                                        AsnGlnGluAspAlaGlu AlaIleThrHisLeuPheThrAspValGln                             100105110                                                                     AsnArgTyrThrPheAlaPheGlyGlyAsnTyrAspArgLeuGluGln                              115 120125                                                                    LeuAlaGlyAsnLeuArgGluAsnIleGluLeuGlyAsnGlyProLeu                              130135140                                                                     GluGluAlaIleSerAlaLeuTyrTyrT yrSerThrGlyGlyThrGln                             145150155160                                                                  LeuProThrLeuAlaArgSerPheIleIleCysIleGlnMetIleSer                              165 170175                                                                    GluAlaAlaArgPheGlnTyrIleGluGlyGluMetArgThrArgIle                              180185190                                                                     ArgTyrAsnArgArgSerAl aProAspProSerValIleThrLeuGlu                             195200205                                                                     AsnSerTrpGlyArgLeuSerThrAlaIleGlnGluSerAsnGlnGly                              210215 220                                                                    AlaPheAlaSerProIleGlnLeuGlnArgArgAsnGlySerLysPhe                              225230235240                                                                  SerValTyrAspValSerIleLeu IleProIleIleAlaLeuMetVal                             245250255                                                                     TyrArgCysAlaProProProSerSerGlnPhe                                             260265                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 251 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GlyLeuAspThrValSerPheSerThrLysGlyAlaThrTyrIleThr                              15 1015                                                                       TyrValAsnPheLeuAsnGluLeuArgValLysLeuLysProGluGly                              202530                                                                        AsnSerHisGlyIlePro LeuLeuArgLysLysCysAspAspProGly                             354045                                                                        LysCysPheValLeuValAlaLeuSerAsnAspAsnGlyGlnLeuAla                              5055 60                                                                       GluIleAlaIleAspValThrSerValTyrValValGlyTyrGlnVal                              65707580                                                                      ArgAsnArgSerTyrPhePheLys AspAlaProAspAlaAlaTyrGlu                             859095                                                                        GlyLeuPheLysAsnThrIleLysThrArgLeuHisPheGlyGlyThr                              100 105110                                                                    TyrProSerLeuGluGlyGluLysAlaTyrArgGluThrThrAspLeu                              115120125                                                                     GlyIleGluProLeuArgIleGlyIl eLysLysLeuAspGluAsnAla                             130135140                                                                     IleAspAsnTyrLysProThrGluIleAlaSerSerLeuLeuValVal                              145150 155160                                                                 IleGlnMetValSerGluAlaAlaArgPheThrPheIleGluAsnGln                              165170175                                                                     IleArgAsnAsnPheGlnGlnArg IleArgProAlaAsnAsnThrIle                             180185190                                                                     SerLeuGluAsnLysTrpGlyLysLeuSerPheGlnIleArgThrSer                              195 200205                                                                    GlyAlaAsnGlyMetPheSerGluAlaValGluLeuGluArgAlaAsn                              210215220                                                                     GlyLysLysTyrTyrValThrAlaValAspGln ValLysProLysIle                             225230235240                                                                  AlaLeuLeuLysPheValAspLysAspProLys                                             245250                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                               (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 280 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AlaAlaLysMetAlaLysAsnValAspLysProLeuPheThrAlaThr                              15 1015                                                                       PheAsnValGlnAlaSerSerAlaAspTyrAlaThrPheIleAlaGly                              202530                                                                        IleArgAsnLysLeuArgAsnP roAlaHisPheSerHisAsnArgPro                             354045                                                                        ValLeuProProValGluProAsnValProProSerArgTrpPheHis                              5055 60                                                                       ValValLeuLysAlaSerProThrSerAlaGlyLeuThrLeuAlaIle                              65707580                                                                      ArgAlaAspAsnIleTyrLeuGluGlyP heLysSerSerAspGlyThr                             859095                                                                        TrpTrpGluLeuThrProGlyLeuIleProGlyAlaThrTyrValGly                              100 105110                                                                    PheGlyGlyThrTyrArgAspLeuLeuGlyAspThrAspLysLeuThr                              115120125                                                                     AsnValAlaLeuGlyArgGlnGlnLeuAla AspAlaValThrAlaLeu                             130135140                                                                     HisGlyArgThrLysAlaAspLysAlaSerGlyProLysGlnGlnGln                              145150155 160                                                                 AlaArgGluAlaValThrThrLeuValLeuMetValAsnGluAlaThr                              165170175                                                                     ArgPheGlnThrValSerGlyPheVal AlaGlyLeuLeuHisProLys                             180185190                                                                     AlaValGluLysLysSerGlyLysIleGlyAsnGluMetLysAlaGln                              195200 205                                                                    ValAsnGlyTrpGlnAspLeuSerAlaAlaLeuLeuLysThrAspVal                              210215220                                                                     LysProProProGlyLysSerProAlaLysPheAlaP roIleGluLys                             225230235240                                                                  MetGlyValArgThrAlaGluGlnAlaAlaAsnThrLeuGlyIleLeu                              24525 0255                                                                    LeuPheValGluValProGlyGlyLeuThrValAlaLysAlaLeuGlu                              260265270                                                                     LeuPheHisAlaSerGlyGlyLys                                                       275280                                                                       (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 263 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       AspValAsnPheAspLeuSerThrAlaThrAlaLysThr TyrThrLys                             151015                                                                        PheIleGluAspPheArgAlaThrLeuProPheSerHisLysValTyr                              2025 30                                                                       AspIleProLeuLeuTyrSerThrIleSerAspSerArgArgPheIle                              354045                                                                        LeuLeuAspLeuThrSerTyrAlaTyrGluThrIleSerVal AlaIle                             505560                                                                        AspValThrAsnValTyrValValAlaTyrArgThrArgAspValSer                              65707580                                                                      TyrPhePheLysGluSerProProGluAlaTyrAsnIleLeuPheLys                              859095                                                                        GlyThrArgLysIleThrLeuProTyrThrGlyAsnTyrGlu AsnLeu                             100105110                                                                     GlnThrAlaAlaHisLysIleArgGluAsnIleAspLeuGlyLeuPro                              115120125                                                                      AlaLeuSerSerAlaIleThrThrLeuPheTyrTyrAsnAlaGlnSer                             130135140                                                                     AlaProSerAlaLeuLeuValLeuIleGlnThrThrAlaGluAlaAla                               145150155160                                                                 ArgPheLysTyrIleGluArgHisValAlaLysTyrValAlaThrAsn                              16517017 5                                                                    PheLysProAsnLeuAlaIleIleSerLeuGluAsnGlnTrpSerAla                              180185190                                                                     LeuSerLysGlnIlePheLeuAlaGlnAsnGlnGlyGlyLysPh eArg                             195200205                                                                     AsnProValAspLeuIleLysProThrGlyGluArgPheGlnValThr                              210215220                                                                     Asn ValAspSerAspValValLysGlyAsnIleLysLeuLeuLeuAsn                             225230235240                                                                  SerArgAlaSerThrAlaAspGluAsnPheIleThrThrMetThrLeu                              245250255                                                                     LeuGlyGluSerValValAsn                                                         260                                                                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 248 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       AspValArgPheSerLeuSerGlySerSerSerThrSerTyrSerLys                              151015                                                                        PheIleGlyAspLeuArg LysAlaLeuProSerAsnGlyThrValTyr                             202530                                                                        AsnLeuThrIleLeuLeuSerSerAlaSerGlyAlaSerArgTyrThr                              35 4045                                                                       LeuMetThrLeuSerAsnTyrAspGlyLysAlaIleThrValAlaVal                              505560                                                                        AspValSerGlnLeuTyrIleMetGlyTyr LeuValAsnSerThrSer                             65707580                                                                      TyrPhePheAsnGluSerAspAlaLysLeuAlaSerGlnTyrValPhe                              85 9095                                                                       LysGlySerThrIleValThrLeuProTyrSerGlyAsnTyrGluLys                              100105110                                                                     LeuGlnThrAlaAlaGlyLysIle ArgGluLysIleProLeuGlyPhe                             115120125                                                                     ProAlaLeuAspSerAlaLeuThrThrIlePheHisTyrAspSerThr                              130135 140                                                                    AlaAlaAlaAlaAlaPheLeuValIleLeuGlnThrThrAlaGluAla                              145150155160                                                                  SerArgPheLysTyrIleGluGlyGln IleIleGluArgIleSerLys                             165170175                                                                     AsnGlnValProSerLeuAlaThrIleSerLeuGluAsnSerLeuTrp                              180 185190                                                                    SerAlaLeuSerLysGlnIleGlnLeuAlaGlnThrAsnAsnGlyThr                              195200205                                                                     PheLysThrProValValIleThrAspA spLysGlyGlnArgValGlu                             210215220                                                                     IleThrAsnValThrSerLysValValThrLysAsnIleGlnLeuLeu                              22523023 5240                                                                 LeuAsnTyrLysGlnAsnValAla                                                      245                                                                           (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 255 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                        AspValSerPheArgLeuSerGlyAlaThrSerSerSerTyrGlyVal                             151015                                                                        PheIleSerAsnLeuArgLysAlaLeuProAsnGluArgLysLeuT yr                             202530                                                                        AspLeuProLeuIleArgSerSerLeuProGlySerGlnArgTyrAla                              354045                                                                        I leIleHisLeuThrAsnTyrAlaAspGluValAlaLeuAspValThr                             505560                                                                        AsnValAspAlaGlyLeuProArgAsnAlaValLeuTyrIleMetGly                              65 707580                                                                     TyrArgAlaGlyAspThrSerTyrPhePheAsnGluAlaSerAlaThr                              859095                                                                        G luAlaAlaLysTyrValPheLysAspAlaMetArgLysValThrLeu                             100105110                                                                     ProTyrSerGlyAsnTyrGluArgLeuGlnThrAlaAlaGlyGlyLeu                               115120125                                                                    ArgGluAsnIleProLeuGlyLeuProAlaLeuAspSerAlaIleThr                              130135140                                                                     ThrLeuPheTy rTyrAsnAlaAsnSerAlaAlaSerAlaLeuMetVal                             145150155160                                                                  LeuIleGlnSerThrSerGluAlaAlaArgTyrLysPheIleGluGln                               165170175                                                                    GlnIleGlySerArgValAspLysThrPheLeuProSerLeuAlaIle                              180185190                                                                     Ile SerLeuGluAsnSerLeuTrpLeuAlaLeuSerLysGlnIleGln                             195200205                                                                     IleAlaSerThrAsnAsnGlyGluPheGluThrProValValLeuIle                               210215220                                                                    AsnAlaGlnAsnGlnArgValThrIleThrAsnValAspAlaGlyVal                              225230235240                                                                  ValThr SerAsnIleAlaLeuLeuLeuAsnArgAsnAsnMetAla                                245250255                                                                     (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 263 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       AspValSerPheArgLeuSerGlyAlaAspProArgSerTyrGlyMet                              151015                                                                        PheIleLysAspLeuArgAsnAl aLeuProPheArgGluLysValTyr                             202530                                                                        AsnIleProLeuLeuLeuProSerValSerGlyAlaGlyArgTyrLeu                              35 4045                                                                       LeuMetHisLeuPheAsnTyrAspGlyLysThrIleThrValAlaVal                              505560                                                                        AspValThrAsnValTyrIleMetGlyTyrLeuAl aAspThrThrSer                             65707580                                                                      TyrPhePheAsnGluProAlaAlaGluLeuAlaSerGlnTyrValPhe                              85 9095                                                                       ArgAspAlaArgArgLysIleThrLeuProTyrSerGlyAsnTyrGlu                              100105110                                                                     ArgLeuGlnIleAlaAlaGlyLysProA rgGluLysIleProIleGly                             115120125                                                                     LeuProAlaLeuAspSerAlaIleSerThrLeuLeuHisTyrAspSer                              130135 140                                                                    ThrAlaAlaAlaGlyAlaLeuLeuValLeuIleGlnThrThrAlaGlu                              145150155160                                                                  AlaAlaArgPheLysTyrIleGluGlnGlnIl eGlnGluArgAlaTyr                             165170175                                                                     ArgAspGluValProSerLeuAlaThrIleSerLeuGluAsnSerTrp                              180185 190                                                                    SerGlyLeuSerLysGlnIleGlnLeuAlaGlnGlyAsnAsnGlyIle                              195200205                                                                     PheArgThrProIleValLeuValAspAsnLys GlyAsnArgValGln                             210215220                                                                     IleThrAsnValThrSerLysValValThrSerAsnIleGlnLeuLeu                              225230235 240                                                                 LeuAsnThrArgAsnIleAlaGluGlyAspAsnGlyAspValSerThr                              245250255                                                                     ThrHisGlyPheSerSerThr                                                          260                                                                          (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 250 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       AlaProThrLeuGluThrIleAlaSerLeuAspLeuAsnAsnProThr                              1 51015                                                                       ThrTyrLeuSerPheIleThrAsnIleArgThrLysValAlaAspLys                              202530                                                                        ThrGl uGlnCysThrIleGlnLysIleSerLysThrPheThrGlnArg                             354045                                                                        TyrSerTyrIleAspLeuIleValSerSerThrGlnLysIleThrLeu                              50 5560                                                                       AlaIleAspMetAlaAspLeuTyrValLeuGlyTyrSerAspIleAla                              65707580                                                                      AsnAsnLysGl yArgAlaPhePhePheLysAspValThrGluAlaVal                             859095                                                                        AlaAsnAsnPhePheProGlyAlaThrGlyThrAsnArgIleLysLeu                               100105110                                                                    ThrPheThrGlySerTyrGlyAspLeuGluLysAsnGlyGlyLeuArg                              115120125                                                                     LysAspAsnPro LeuGlyIlePheArgLeuGluAsnSerIleValAsn                             130135140                                                                     IleTyrGlyLysAlaGlyAspValLysLysGlnAlaLysPhePheLeu                              1451 50155160                                                                 LeuAlaIleGlnMetValSerGluAlaAlaArgPheLysTyrIleSer                              165170175                                                                     AspLysIleP roSerGluLysTyrGluGluValThrValAspGluTyr                             180185190                                                                     MetThrAlaLeuGluAsnAsnTrpAlaLysLeuSerThrAlaValTyr                              19 5200205                                                                    AsnSerLysProSerThrThrThrAlaThrLysCysGlnLeuAlaThr                              210215220                                                                     SerProValThrIleSerPr oTrpIlePheLysThrValGluGluIle                             225230235240                                                                  LysLeuValMetGlyLeuLeuLysSerSer                                                245250                                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 261 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       IleAsnThrIleThrPheAspAlaGlyAsnAlaThrIleAsnLysTyr                              1 51015                                                                       AlaThrPheMetGluSerLeuArgAsnGluAlaLysAspProSerLeu                              202530                                                                        LysCysTyrGly IleProMetLeuProAsnThrAsnSerThrIleLys                             354045                                                                        TyrLeuLeuValLysLeuGlnGlyAlaSerLeuLysThrIleThrLeu                              50 5560                                                                       MetLeuArgArgAsnAsnLeuTyrValMetGlyTyrSerAspProTyr                              65707580                                                                      AspAsnLysCysArgTyr HisIlePheAsnAspIleLysGlyThrGlu                             859095                                                                        TyrSerAspValGluAsnThrLeuCysProSerSerAsnProArgVal                              100 105110                                                                    AlaLysProIleAsnTyrAsnGlyLeuTyrProThrLeuGluLysLys                              115120125                                                                     AlaGlyValThrSerArgA snGluValGlnLeuGlyIleGlnIleLeu                             130135140                                                                     SerSerLysIleGlyLysIleSerGlyGlnGlySerPheThrGluLys                              145150 155160                                                                 IleGluAlaAspPheLeuLeuValAlaIleGlnMetValSerGluAla                              165170175                                                                     AlaArgPheLysTyrIl eGluAsnGlnValLysThrAsnPheAsnArg                             180185190                                                                     AspPheSerProAsnAspLysValLeuAspLeuGluGluAsnTrpGly                              195 200205                                                                    LysIleSerThrAlaIleHisAsnSerLysAsnGlyAlaLeuProLys                              210215220                                                                     ProLeuGluLeuLysAsnAlaAspGly ThrLysTrpIleValLeuArg                             225230235240                                                                  ValAspGluIleLysProAspValGlyLeuLeuAsnTyrValAsnGly                              245 250255                                                                    ThrCysGlnAlaThr                                                               260                                                                           (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 259 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                       ValThrSerIleThrLeuAspLeuValAsnProThrAlaGlyGlnTyr                             151015                                                                        SerSerPheValAspLysIleArgAsnAsnValLysAspProAsnLeu                              202530                                                                        LysTyrGlyGlyThrAspIleAlaValIleGlyProProSerLysGlu                              354045                                                                        Lys PheLeuArgIleAsnPheGlnSerSerArgGlyThrValSerLeu                             505560                                                                        GlyLeuLysArgAspAsnLeuTyrValValAlaTyrLeuAlaMetAsp                              65 707580                                                                     AsnThrAsnValAsnArgAlaTyrTyrPheArgSerGluIleThrSer                              859095                                                                        Ala GluSerThrAlaLeuPheProGluAlaThrThrAlaAsnGlnLys                             100105110                                                                     AlaLeuGluTyrThrGluAspTyrGlnSerIleGluLysAsnAlaGln                               115120125                                                                    IleThrGlnGlyAspGlnSerArgLysGluLeuGlyLeuGlyIleAsp                              130135140                                                                     LeuLeuSerThr SerMetGluAlaValAsnLysLysAlaArgValVal                             145150155160                                                                  LysAspGluAlaArgPheLeuLeuIleAlaIleGlnMetThrAlaGlu                               165170175                                                                    AlaAlaArgPheArgTyrIleGlnAsnLeuValIleLysAsnPhePro                              180185190                                                                     AsnL ysPheAsnSerGluAsnLysValIleGlnPheGluValAsnTrp                             195200205                                                                     LysLysIleSerThrAlaIleTyrGlyAspAlaLysAsnGlyValPhe                              21 0215220                                                                    AsnLysAspTyrAspPheGlyPheGlyLysValArgGlnValLysAsp                              225230235240                                                                  LeuGlnMe tGlyLeuLeuMetTyrLeuGlyLysProLysSerSerAsn                             245250255                                                                     GluAlaAsn                                                                     (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 813 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GGGCTAGATACCGTGTCATTCTCAACCAAAGGTGCCACTTATATTACCTACGTGAATTTC60                TTGAATGAGCTACGAGTTAAATTGAAACCCGAAGGTAACAGCCATGGAATCCC ATTGCTG120              CGCAAAAAATGTGATGATCCTGGAAAGTGTTTCGTTTTGGTAGCGCTTTCAAATGACAAT180               GGACAGTTGGCGGAAATAGCTATAGATGTTACAAGTGTTTATGTGGTGGGCTATCAAGTA240               AGAAACAGATCTTACTTCTTTAAAGATGCT CCAGATGCTGCTTACGAAGGCCTCTTCAAA300              AACACAATTAAAACAAGACTTCATTTTGGCGGCACGTATCCCTCGCTGGAAGGTGAGAAG360               GCATATAGAGAGACAACAGACTTGGGCATTGAACCATTAAGGATTGGCATCAAGAAACTT420               GATGAAAA TGCGATAGACAATTATAAACCAACGGAGATAGCTAGTTCTCTATTGGTTGTT480              ATTCAAATGGTGTCTGAAGCAGCTCGATTCACCTTTATTGAGAACCAAATTAGAAATAAC540               TTTCAACAGAGAATTCGCCCGGCGAATAATACAATCAGCCTTGAGAATAAATG GGGTAAA600              CTCTCGTTCCAGATCCGGACATCAGGTGCAAATGGAATGTTTTCGGAGGCAGTTGAATTG660               GAACGTGCAAATGGCAAAAAATACTATGTCACCGCAGTTGATCAAGTAAAACCCAAAATA720               GCACTCTTGAAGTTCGTCGATAAAGATCCT AAAACGAGCCTTGCTGCTGAATTGATAATC780              CAGAACTATGAGTCATTAGTGGGCTTTGATTAG813                                          (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 846 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      ATGGCGGCAAAGATGGCGAAGAACGTGGACAAGCCGCTCTTCACCGCGACGTTCAACGTC60                CAGGCCAGCTCCGCCGACTACGCCACCTTCATCGCCGGCATCCGCAACAAGCTCCGCAAC120               CCGGCGC ACTTCTCCCACAACCGCCCCGTGCTGCCGCCGGTCGAGCCCAACGTCCCGCCG180              AGCAGGTGGTTCCACGTCGTGCTCAAGGCCTCGCCGACCAGCGCCGGGCTCACGCTGGCC240               ATCCGCGCGGACAACATCTACCTGGAGGGCTTCAAGAGCAGCGACGGCACCT GGTGGGAG300              CTCACCCCGGGCCTCATCCCCGGCGCCACCTACGTCGGGTTCGGCGGCACCTACCGCGAC360               CTCCTCGGCGACACCGACAAGCTAACCAACGTCGCTCTCGGCCGACAGCAGCTGGCGGAC420               GCGGTGACCGCGCTCCACGGGCGCACCAAG GCCGACAAGGCCTCCGGCCCGAAGCAGCAG480              CAGGCGAGGGAGGCGGTGACGACGCTGGTCCTCATGGTGAACGAGGCCACGCGGTTCCAG540               ACGGTGTCTGGGTTCGTGGCCGGGTTGCTGCACCCCAAGGCGGTGGAGAAGAAGAGCGGG600               AAGATCG GCAATGAGATGAAGGCCCAGGTGAACGGGTGGCAGGACCTGTCCGCGGCGCTG660              CTGAAGACGGACGTGAAGCCTCCGCCGGGAAAGTCGCCAGCGAAGTTCGCGCCGATCGAG720               AAGATGGGCGTGAGGACGGCTGAACAGGCCGCCAACACGCTGGGGATCCTGC TGTTCGTG780              GAGGTGCCGGGTGGGTTGACGGTGGCCAAGGCGCTGGAGCTGTTCCATGCGAGTGGTGGG840               AAATAG846                                                                     (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 913 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      CGTCCGAAAATGGTGAAATGCTTACTACTTTCTTTTTTAATTATCGCCATCTTCATTGGT60                GTTCCTACTGCCAAAGGCGATGTTAACTT CGATTTGTCGACTGCCACTGCAAAAACCTAC120              ACAAAATTTATCGAAGATTTCAGGGCGACTCTTCCATTTAGCCATAAAGTGTATGATATA180               CCTCTACTGTATTCCACTATTTCCGACTCCAGACGTTTCATACTCCTCGATCTTACAAGT240               TATGCA TATGAAACCATCTCGGTGGCCATAGATGTGACGAACGTTTATGTTGTGGCGTAT300              CGCACCCGCGATGTATCCTACTTTTTTAAAGAATCTCCTCCTGAAGCTTATAACATCCTA360               TTCAAAGGTACGCGGAAAATTACACTGCCATATACCGGTAATTATGAAAAT CTTCAAACT420              GCTGCACACAAAATAAGAGAGAATATTGATCTTGGACTCCCTGCCTTGAGTAGTGCCATT480               ACCACATTGTTTTATTACAATGCCCAATCTGCTCCTTCTGCATTGCTTGTACTAATCCAG540               ACGACTGCAGAAGCTGCAAGATTTAAGTA TATCGAGCGACACGTTGCTAAGTATGTTGCC600              ACTAACTTTAAGCCAAATCTAGCCATCATAAGCTTGGAAAATCAATGGTCTGCTCTCTCC660               AACAAATCTTTTTGGCGCAGAATCAAGGAGGAAAATTTAGAAATCCTGTCGACCTTATAA720               AACCTA CCGGGGAACGGTTTCAAGTAACCAATGTTGATTCAGATGTTGTAAAAGGTAATA780              TCAAACTCCTGCTGAACTCCAGAGCTAGCACTGCTGATGAAAACTTTATCACAACCATGA840               CTCTACTTGGGGAATCTGTTGTGAATTGAAAGTTTAATAATCCACCCATAT CGAAATAAG900              GCATGTTCATGAC913                                                              (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      TTYAARGAYGCNCCNGAYGCNGCNTAYGARGG32                                            (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      ACYTGRTCNACNGCNGTNACRTARTAYTTYTT32                                            (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GGNYTNGAYACNGTNWSNTTYWSNACNAARGG32                                            (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii ) MOLECULE TYPE: DNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      AATGGTTCAATGCCCAAGTCTGT23                                                     (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (i i) MOLECULE TYPE: DNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      TGTCTCTCTATATGCCTTTCCAC23                                                     (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 53 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          ( ii) MOLECULE TYPE: DNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      TCAACCCGGGCTAGATACCGTGTCATTCTCAACCAAAGGTGCCACTTATATTA53                       (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      CTTCATTTTGGCGGCACGTATCC23                                                     (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      CTCGAGGCTGCAAGCTTACGTGGGATTTTTTTTTTTTTTTTTTTTT46                              (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      CTCGCTGGAAGGTGAGAA18                                                          (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      CTCGAGGCTGCAAGCTTACGTGGGA25                                                   (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      TGATCTCGAGTACTATTTAGGATCTTTATCGACGA35                                         (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      GTAAGCAGCATCTGGAGCATCT22                                                      (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      CATTCAAGAAATTCACGTAGG21                                                       (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      GGCCTGGACACCGTGAGCTTTAG23                                                     (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      TCGATTGCGATCCTAAATAGTACTC25                                                   (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      TTTAGGATCGCAATCGACGAACTTCAAG28                                                (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D ) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      GTTCGTCTGTAAAGATCCTAAATAGTACTCGA32                                            (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      ( D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      GGATCTTTACAGACGAACTTCAAGAGT27                                                 (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      TCTTGTGCTTCGTCGATAAAGATCC25                                                   (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      ATCGACGAAGCACAAGAGTGCTATTTT27                                                 (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      GTAAAACCATGCATAGCACTCTTGAAGTTCGT32                                            (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      AGTGCTATGCATGGTTTTACTTGATCAACTGC32                                            (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      AGCACATGTGGTGCCACTTATATTACCTA29                                               (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      TAAGTGGCACCACATGTGCTAAAGCTCACGGTG33                                           (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      TGACTGTGGACAGTTGGCGGAAATA25                                                   (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      GCCACTGTCCACAGTCATTTGAAAGCGCTACC32                                            (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      GATGATCCTGGAAAGGCTTTCGTTTTGGTAGCGCTT36                                        (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      AAGCCTTTCCAGGATCATCAGCTTTTTTGGGCAGCAATGGG41                                   (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      AAGCCTTTCCAGGATCATCACAT23                                                     (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      GCGACTCTCTACTGTTTC18                                                          (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      CGTTAGCAATTTAACTGTGAT21                                                       (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      AACAGCTATGACCATG16                                                            (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      TGAACTCGAGGAAACTACCTATTTCCCAC29                                               (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C ) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      GCATTACATCCATGGCGGC19                                                         (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 64 base pairs                                                     (B) TYPE: nucleic acid                                                        ( C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      GATATCTCGAGTTAACTATTTCCCACCACACGCATGGAACAGCTCCAGCGCCTTGGCCAC60                CGTC6 4                                                                       (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      TATCTGTTCGTGGAGGTGCCG 21                                                      (2) INFORMATION FOR SEQ ID NO:50:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                      CGTTAGCAATTTAACTGTGAT 21                                                      (2) INFORMATION FOR SEQ ID NO:51:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                      CCAAGTGTCTGGAGCTGTTCCATGCGA 27                                                (2) INFORMATION FOR SEQ ID NO:52:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                      GATGTTAAYTTYGAYTTGTCNACDGCTAC 29                                              (2) INFORMATION FOR SEQ ID NO:53:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                      ATTGGNAGDGTAGCCCTRAARTCYTCDAT 29                                              (2) INFORMATION FOR SEQ ID NO:54:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                      GCCACTGCAAAAACCTACACAAAATTTATTGA 32                                           (2) INFORMATION FOR SEQ ID NO:55:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                      GATGTTAACTTCGATTTGTCGA 22                                                     (2) INFORMATION FOR SEQ ID NO:56:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                      TCAACTCGAGGTACTCAATTCACAACAGATTCC 33                                          (2) INFORMATION FOR SEQ ID NO:57:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 813 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                      GGCCTGGACACCGTGAGCTTTAGCACTAAAGGTGCCACTTATATTACCTACGTGAAT TTC60               TTGAATGAGCTACGAGTTAAATTGAAACCCGAAGGTAACAGCCATGGAATCCCATTGCTG120               CGCAAAAAATGTGATGATCCTGGAAAGTGTTTCGTTTTGGTAGCGCTTTCAAATGACAAT180               GGACAGTTGGCGGAAATAGCTATAGATGTTACAA GTGTTTATGTGGTGGGCTATCAAGTA240              AGAAACAGATCTTACTTCTTTAAAGATGCTCCAGATGCTGCTTACGAAGGCCTCTTCAAA300               AACACAATTAAAACAAGACTTCATTTTGGCGGCACGTATCCCTCGCTGGAAGGTGAGAAG360               GCATATAGAGA GACAACAGACTTGGGCATTGAACCATTAAGGATTGGCATCAAGAAACTT420              GATGAAAATGCGATAGACAATTATAAACCAACGGAGATAGCTAGTTCTCTATTGGTTGTT480               ATTCAAATGGTGTCTGAAGCAGCTCGATTCACCTTTATTGAGAACCAAATTAGAAAT AAC540              TTTCAACAGAGAATTCGCCCGGCGAATAATACAATCAGCCTTGAGAATAAATGGGGTAAA600               CTCTCGTTCCAGATCCGGACATCAGGTGCAAATGGAATGTTTTCGGAGGCAGTTGAATTG660               GAACGTGCAAATGGCAAAAAATACTATGTCACCG CAGTTGATCAAGTAAAACCCAAAATA720              GCACTCTTGAAGTTCGTCGATAAAGATCCTAAAACGAGCCTTGCTGCTGAATTGATAATC780               CAGAACTATGAGTCATTAGTGGGCTTTGATTAG813                                      

We claim:
 1. A non-naturally occurring analog of the Type Iribosome-inactivating protein, gelonin, wherein a cysteine issubstituted for another amino acid at an amino acid position notnaturally available for intermolecular disulfide bonding in said geloninand said cysteine being available for intermolecular disulfide bonding,wherein said cysteine is substituted at an amino acid position in saidgelonin from position 239 to the carboxy terminus, wherein said analogretains ribosome-inactivating activity of said gelonin, and wherein saidgelonin comprises the amino acid sequence shown in SEQ ID NO:
 2. 2. Thegelonin analog recited in claim 1 wherein said cysteine is substitutedat a position selected from the group consisting of positions 239, 244,247, and 248 of the amino sequence of said analog.
 3. The gelonin analogrecited in claim 1 or 2 wherein additionally the native gelonin cysteineresidues at positions 44 and 50 are replaced with alanine residues.
 4. Anon-naturally occurring analog of barley ribosome-inactivating proteinwherein a cysteine is substituted for another amino acid at an aminoacid position not naturally available for intermolecular disulfidebonding in said barley ribosome-inactivating protein and said cysteinebeing available for intermolecular disulfide bonding and wherein saidcysteine is substituted at an amino acid position in said barleyribsome-inactivating protein from position 256 to the carboxy terminus,wherein said analog retains ribosome-inactivating of said barleyribosome-inactivating protein, and wherein said barleyribosome-inactivating protein comprises the amino acid sequence shown inSEQ ID NO:
 3. 5. The barley ribosome-inactivating protein analog recitedin claim 4 wherein said cysteine is at position 256 of the amino acidsequence of said analog.
 6. The barley ribosome-inactivating proteinanalog recited in claim 4 wherein said cysteine is substituted atposition 270 of the amino acid sequence of said analog.
 7. The barleyribosome-inactivating protein analog recited in claim 4 wherein saidcysteine is substituted at position 277 of the amino acid of saidanalog.
 8. An analog of the Type I ribosome-inactivating protein,gelonin, comprising a cysteine residue substituted for a non-cysteineresidue at position 10 in the amino acid sequence shown in SEQ ID NO: 2.9. An analog of the Type I ribosome-inactivating protein, gelonin,comprising a cysteine residue substituted for a non-cysteine residue atposition 60 in the amino acid sequence shown in SEQ ID NO:
 2. 10. Ananalog of the Type I ribosome-inactivating protein, gelonin, comprisinga cysteine residue substituted for a non-cysteine residue at position 50in the amino acid sequence shown in SEQ ID NO:
 2. 11. An analog of theType I ribosome-inactivating protein, gelonin, comprising a cysteineresidue substituted for a non-cysteine residue at position 44 in theamino acid sequence shown in SEQ ID NO: 2.